Alpha polyglutamated tetrahydrofolates and uses thereof

ABSTRACT

The disclosure relates generally to polyglutamated alpha tetrahydrofolate compositions, including delivery vehicles such as liposomes containing the polyglutamated alpha tetrahydrofolate, and methods of making and using the polyglutamated alpha tetrahydrofolate compositions to treat hyperproliferative disorders (e.g., cancer) and disorders of the immune system (e.g., inflammation and autoimmune diseases such as rheumatoid arthritis). The disclosed compositions also have uses in combination therapy with one or more therapeutic agents to enhance the effectiveness or to reduce the toxicities associated with the therapeutic agent(s).

BACKGROUND

This disclosure generally relates to polyglutamated alphatetrahydrofolate compositions, including delivery vehicles such asliposomes containing the polyglutamated alpha tetrahydrofolatecompositions, and methods of making and using the compositions to treatdiseases including hyperproliferative diseases such as cancer, disordersof the immune system such as rheumatoid arthritis, infectious diseasessuch as HIV and malaria. The polyglutamated alpha tetrahydrofolatecompositions also have uses in combination therapy with one or moretherapeutic agents such as a chemotherapeutic drug (e.g.,5-fluorouracil) to enhance the effectiveness of the therapeutic agent(s)or as a “chemoprotectant” (e.g., in combination with antifolates such asmethotrexate) to reduce toxic side effects associated with thetherapeutic agent(s).

Folate is an essential cofactor that mediates the transfer of one-carbonunits involved in nucleotide biosynthesis and DNA repair, theremethylation of homocysteine (Hcy), and the methylation of DNA,proteins, and lipids. The only circulating forms of folates in the bloodare monoglutamates and folate monoglutamates are the only form of folatethat is transported across the cell membrane—likewise, the monoglutamateform of tetrahydrofolate, are transported across the cell membrane. Oncetaken up into cells, intracellular tetrahydrofolate is polyglutamated bythe enzyme folylpoly-gamma-glutamate synthetase (FPGS). Thepolyglutamation of tetrahydrofolate by FPGS serves at least 2 maintherapeutic purposes: (1) it greatly enhances tetrahydrofolate affinityfor DHFR; and (2) it facilitates the accumulation of polyglutamatedtetrahydrofolate, which unlike tetrahydrofolate (monoglutamate), is noteasily transported out of cells by cell efflux pumps.

The provided polyglutamated alpha tetrahydrofolate compositions delivera strategy for improving the therapeutic efficacy of tetrahydrofolate.

BRIEF SUMMARY

This disclosure generally relates polyglutamated alpha tetrahydrofolate(THF) compositions and methods of making and using the compositions totreat diseases including hyperproliferative diseases such as cancer,disorders of the immune system such as inflammation and rheumatoidarthritis, and infectious disease such as HIV and malaria. Thepolyglutamated alpha tetrahydrofolate compositions also have uses incombination therapy with one or more therapeutic agents such as achemotherapeutic drug (e.g., 5-fluorouracil) to enhance theeffectiveness of the therapeutic agent(s) or as a “chemoprotectant”(e.g., in combination with antifolates such as methotrexate) to reducetoxic side effects associated with the therapeutic agent(s).

In some embodiments, the disclosure provides:

-   -   [1] a composition comprising a polyglutamated alpha        tetrahydrofolate.    -   [2] the composition of [1], wherein the polyglutamated alpha        tetrahydrofolate is selected from the group consisting of:        -   (a) polyglutamated 5-formyl-THF (e.g., polyglutamated            [6S]-5-formyl-THF);        -   (b) polyglutamated 10-formyl-THF (e.g., polyglutamated            [6R]-10-formyl-THF);        -   (c) polyglutamated 5,10-methenyl-THF (e.g., polyglutamated            [6R]-5,10-methenyl-THF);        -   (d) polyglutamated 5-methyl-THF (e.g., polyglutamated            [6S]-5-methyl-THF);        -   (e) polyglutamated tetrahydrofolate (e.g., polyglutamated            [6S]-Tetrahydrofolate THF);        -   (f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated            [6R]-5,10-methylene-THF); and        -   (g) polyglutamated 5-formimino-THF (e.g., polyglutamated            [6S]-5-formimino-THF).    -   [3] the composition of [1] or [2], wherein the polyglutamated        alpha tetrahydrofolate contains 4, 5, 2-10, 4-6, or more than 5,        glutamyl groups having alpha carboxyl group linkages.    -   [4] the composition according to any of [1]-[3], wherein the        polyglutamated alpha tetrahydrofolate is tetraglutamated alpha        tetrahydrofolate (e.g., [6R]-5,10-methenyl-THF,        [6S]-5-formyl-THF and/or [6R]-10-formyl-THF).    -   [5] the composition according to any of [1]-[3], wherein the        polyglutamated alpha tetrahydrofolate is pentaglutamated alpha        tetrahydrofolate (e.g., [6R]-5,10-methenyl-THF,        [6S]-5-formyl-THF and/or [6R]-10-formyl-THF).    -   [6] the composition according to any of [1]-[3], wherein the        polyglutamated alpha tetrahydrofolate is hexaglutamated alpha        tetrahydrofolate (e.g., [6R]-5,10-methenyl-THF,        [6S]-5-formyl-THF and/or [6R]-10-formyl-THF).    -   [7] the composition according to any of [1] to [6], wherein        -   (a) two or more glutamyl groups have an alpha carboxyl group            linkage,        -   (b) each of the glutamyl groups other than the glutamyl            group of tetrahydrofolate has an alpha carboxyl group            linkage; or        -   (c) two or more glutamyl groups have a gamma carboxyl group            linkage.    -   [8] the composition according to any of [1]-[7], wherein at        least one glutamyl group has both an alpha carboxyl group        linkage and a gamma carboxyl group linkage.    -   [9] the composition according to any of [1]-[8], wherein:        -   (a) at least 2 of the glutamyl groups of the polyglutamated            alpha tetrahydrofolate are in the L-form,        -   (b) each of the glutamyl groups of the polyglutamated alpha            tetrahydrofolate is in the L-form,        -   (c) at least 1 of the glutamyl groups of the polyglutamated            alpha tetrahydrofolate is in the D-form,        -   (d) each of the glutamyl groups of the polyglutamated alpha            tetrahydrofolate other than the glutamyl group of            tetrahydrofolate is in the D-form, or        -   (e) at least 2 of the glutamyl groups of the polyglutamated            alpha tetrahydrofolate are in the L-form and at least 1 of            the glutamyl groups is in the D-form.    -   [10] the composition according to any of [1]-[9], wherein the        polyglutamate is linear.    -   [11] the composition according to any of [1]-[9], wherein the        polyglutamate is branched.    -   [12] a liposomal composition comprising the polyglutamated alpha        tetrahydrofolate according to any of [1]-[11] (Lp-αPTHF);    -   [13] the LαPP composition according to [12], wherein the        polyglutamated alpha tetrahydrofolate comprises glutamyl groups        in the L-form having alpha carboxyl group linkages;    -   [14] the Lp-αPTHF composition according to [12] or [13], wherein        each of the glutamyl groups of the polyglutamated alpha        tetrahydrofolate is in the L-form;    -   [15] the Lp-αPTHF composition of [12] or [13], wherein at least        one of the glutamyl groups of the polyglutamated alpha        tetrahydrofolate is in the D-form;    -   [16] the Lp-αPTHF composition according to any of [12]-[15],        wherein the liposome comprises a polyglutamated alpha        tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 5,        glutamyl groups;    -   [17] the Lp-αPTHF composition according to any of [12]-[16],        wherein at least one of the glutamyl groups of the        polyglutamated alpha tetrahydrofolate has a gamma carboxyl group        linkage;    -   [18] the composition according to any of [12]-[17], wherein at        least one glutamyl group has both an alpha carboxyl group        linkage and a gamma carboxyl group linkage;    -   [19] the composition according to any of [12]-[18], which        contains 2, 3, 4, 5, 2-10, 4-6, or more than 5, glutamyl groups        that have both an alpha carboxyl group linkage and a gamma        carboxyl group linkage;    -   [20] the Lp-αPTHF composition according to any of [12]-[19],        wherein the liposome comprises a polyglutamated alpha        tetrahydrofolate containing tetraglutamated alpha        tetrahydrofolate, pentaglutamated alpha tetrahydrofolate, or        hexaglutamated alpha tetrahydrofolate;    -   [21] the Lp-αPTHF composition according to any of [12]-[19],        wherein the liposome comprises a polyglutamated alpha        tetrahydrofolate containing tetraglutamated alpha        tetrahydrofolate, pentaglutamated alpha tetrahydrofolate, or        hexaglutamated alpha tetrahydrofolate;    -   [22] the Lp-αPTHF composition according to any of [12]-[21],        wherein the polyglutamate is linear or branched;    -   [23] The Lp-αPTHF composition according to any of [12]-[22],        wherein the liposome is pegylated (PαLp-αPTHF);    -   [24] the Lp-αPTHF composition according to any of [12]-[23],        wherein the liposomes comprise at least 1% weight by weight        (w/w) of the polyglutamated alpha tetrahydrofolate or wherein        during the process of preparing the Lp-αPTHF, at least 1% of the        starting material of polyglutamated alpha THF is encapsulated        (entrapped) in the αPTHF;    -   [25] the Lp-αPTHF composition according to any of [12]-[24],        wherein the liposome has a diameter in the range of 20 nm to 500        nm or 20 nm to 200 nm;    -   [26] the Lp-αPTHF composition according to any of [12]-[25],        wherein the liposome has a diameter in the range of 80 nm to 120        nm;    -   [27] the Lp-αPTHF composition according to any of [12]-[26],        wherein the liposome is formed from liposomal components;    -   [28] the Lp-αPTHF composition according to [27], wherein the        liposomal components comprise at least one of an anionic lipid        and a neutral lipid;    -   [29] the Lp-αPTHF composition according to [27] or [28], wherein        the liposomal components comprise at least one selected from the        group consisting of: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC;        HSPC-PEG; cholesterol; cholesterol-PEG; and        cholesterol-maleimide;    -   [30] the Lp-αPTHF composition according to any of [27]-[29],        wherein the liposomal components comprise at least one selected        from the group consisting of: DSPE; DSPE-PEG; DSPE-PEG-FITC;        DSPE-PEG-maleimide; cholesterol; and HSPC;    -   [31] the Lp-αPTHF composition according to any of [27]-[30],        wherein one or more liposomal components further comprises a        steric stabilizer;    -   [32] the Lp-αPTHF composition according to [31], wherein the        steric stabilizer is at least one selected from the group        consisting of polyethylene glycol (PEG); poly-L-lysine (PLL);        monosialoganglioside (GM1); poly(vinyl pyrrolidone) (PVP);        poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline);        poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol;        poly[N-(2-hydroxypropyl) methacrylamide]; amphiphilic        poly-N-vinylpyrrolidones; L-amino-acid-based polymer;        oligoglycerol, copolymer containing polyethylene glycol and        polypropylene oxide, Poloxamer 188, and polyvinyl alcohol;    -   [33] the Lp-αPTHF composition according to [32], wherein the        steric stabilizer is PEG and the PEG has a number average        molecular weight (Mn) of 200 to 5000 daltons;    -   [34] the Lp-αPTHF composition according to any of [12]-[33],        wherein the liposome is anionic or neutral;    -   [35] the Lp-αPTHF composition according to any of [12]-[33],        wherein the liposome has a zeta potential that is less than or        equal to zero;    -   [36] the Lp-αPTHF composition according to any of [12]-[33],        wherein the liposome has a zeta potential that is between 0 to        −150 mV;    -   [37] the Lp-αPTHF composition according to any of [12]-[33],        wherein the liposome has a zeta potential that is between −30 to        −50 mV;    -   [38] the Lp-αPTHF composition according to any of [12]-[33],        wherein the liposome is cationic;    -   [39] the Lp-αPTHF composition according to any of [12]-[38],        wherein the liposome has an interior space comprising the        polyglutamated alpha tetrahydrofolate and an aqueous        pharmaceutically acceptable carrier;    -   [40] the Lp-αPTHF composition of [39], wherein the        pharmaceutically acceptable carrier comprises a tonicity agent        such as dextrose, mannitol, glycerine, potassium chloride,        sodium chloride, at a concentration of greater than 1%;    -   [41] the Lp-αPTHF composition of [39], wherein the aqueous        pharmaceutically acceptable carrier is trehalose;    -   [42] the Lp-αPTHF composition of [41], wherein the        pharmaceutically acceptable carrier comprises 5% to 20% weight        of trehalose;    -   [43] the Lp-αPTHF composition according to any of [39]-[42],        wherein the pharmaceutically acceptable carrier comprises 1% to        15 weight of dextrose;    -   [44] the Lp-αPTHF composition according to any of [39]-[43],        wherein the interior space of the liposome comprises 5% dextrose        suspended in an HEPES buffered solution;    -   [45] the Lp-αPTHF composition according to any of [39]-[44],        wherein the pharmaceutically acceptable carrier comprises a        buffer such as HEPES Buffered Saline (HBS) or similar, at a        concentration of between 1 to 200 mM and a pH of between 2 to 8;    -   [46] the Lp-αPTHF composition according to any of [39]-[45],        wherein the pharmaceutically acceptable carrier comprises a        total concentration of sodium acetate and calcium acetate of        between 50 mM to 500 mM;    -   [47] the Lp-αPTHF composition according to any of [12]-[46],        wherein the interior space of the liposome has a pH of 5-8 or a        pH of 6-7, or any range therein between;    -   [48] the Lp-αPTHF composition according to any of [12]-[47],        wherein the liposome comprises less than 500,000 or less than        200,000 molecules of the polyglutamated alpha tetrahydrofolate;    -   [49] the Lp-αPTHF composition according to any of [12]-[48],        wherein the liposome comprises between 10 to 100,000 molecules        of the polyglutamated alpha tetrahydrofolate, or any range        therein between;    -   [50] the Lp-αPTHF composition according to any of [12]-[49],        which further comprises a targeting moiety and wherein the        targeting moiety has a specific affinity for a surface antigen        on a target cell of interest;    -   [51] the Lp-αPTHF composition according to [50], wherein the        targeting moiety is attached to one or both of a PEG and the        exterior of the liposome, optionally wherein targeting moiety is        attached to one or both of the PEG and the exterior of the        liposome by a covalent bond;    -   [52] the Lp-αPTHF composition of [50] or [51], wherein the        targeting moiety is a polypeptide;    -   [53] the Lp-αPTHF composition according to any of [50]-[52],        wherein the targeting moiety is an antibody or an antigen        binding fragment of an antibody;    -   [54] the Lp-αPTHF composition according to any of [50]-[53],        wherein the targeting moiety binds the surface antigen with an        equilibrium dissociation constant (Kd) in a range of 0.5×10¹⁰ to        10×10⁻⁶ as determined using BIACORE® analysis;    -   [55] The Lp-αPTHF composition according to any of [50]-[55],        wherein the targeting moiety specifically binds one or more        folate receptors selected from the group consisting of: folate        receptor alpha (FR-α), folate receptor beta (FR-β), and folate        receptor delta (FR-δ);    -   [56] the Lp-αPTHF composition according to any of [50]-[56],        wherein the targeting moiety comprises one or more selected from        the group consisting of: an antibody, a humanized antibody, an        antigen binding fragment of an antibody, a single chain        antibody, a single-domain antibody, a bi-specific antibody, a        synthetic antibody, a pegylated antibody, and a multimeric        antibody;    -   [57] the Lp-αPTHF composition according to any of [50]-[56],        wherein each pegylated liposome comprises from 1 to 1000 or        30-200 targeting moieties;    -   [58] the Lp-αPTHF composition according to any of [39]-[57],        further comprising one or more of an immunostimulatory agent, a        detectable marker and a maleimide, wherein the immunostimulatory        agent, the detectable marker or the maleimide is attached to        said PEG or the exterior of the liposome;    -   [59] the Lp-αPTHF composition of [58], wherein immunostimulating        agent is at least one selected from the group consisting of: a        protein immunostimulating agent; a nucleic acid        immunostimulating agent; a chemical immunostimulating agent; a        hapten; and an adjuvant;    -   [60] the Lp-αPTHF composition of [58] or [59], wherein the        immunostimulating agent is at least one selected from the group        consisting of: a fluorescein; a fluorescein isothiocyanate        (FITC); a DNP; a beta glucan; a beta-1,3-glucan; a        beta-1,6-glucan; a resolvin (e.g., a Resolvin D such as        D_(n-6DPA) or D_(n-3DPA), a Resolvin E, or a T series resolvin);        and a Toll-like receptor (TLR) modulating agent such as, an        oxidized low-density lipoprotein (e.g. OXPAC, PGPC), and an        eritoran lipid (e.g., E5564).    -   [61] the Lp-αPTHF composition according to any of [58]-[60],        wherein the immunostimulatory agent and the detectable marker is        the same;    -   [62] the Lp-αPTHF composition according to any of [58]-[61],        further comprising a hapten;    -   [63] the Lp-αPTHF composition of [62], wherein the hapten        comprises one or more of fluorescein or Beta 1, 6-glucan;    -   [64] the Lp-αPTHF composition according to any of [12]-[63],        which further comprises at least one cryoprotectant selected        from the group consisting of mannitol; trehalose; sorbitol; and        sucrose;    -   [65] a targeted composition comprising the composition according        to any of [1]-[64];    -   [66] a non-targeted composition comprising the composition        according to any of [1]-[49];    -   [67] the Lp-αPTHF composition according to any of [12]-[66],        which further comprises carboplatin and/or pembroluzumab    -   [68] a pharmaceutical composition comprising the liposomal        polyglutamated alpha tetrahydrofolate composition according to        any of [12]-[67];    -   [69] a pharmaceutical composition comprising polyglutamated        alpha tetrahydrofolate composition according to any of [1]-[7];    -   [70] the composition of any of [1]-[69], for use in the        treatment of disease;    -   [71] use of the composition of any of [1]-[70], in the        manufacture of a medicament for the treatment of disease and/or        for use in combination therapy with one or more therapeutic        agents such as a chemotherapeutic drug (e.g., 5-fluorouracil) to        enhance the effectiveness of the therapeutic agent(s) or as a        “chemoprotectant” (e.g., in combination with an antifolate such        as methotrexate) to reduce a toxic side effect associated with        the therapeutic agent(s);    -   [72] a method for treating or preventing disease in a subject        needing such treatment or prevention, the method comprising        administering the composition of any of [1]-[70] to the subject;    -   [73] a method for treating or preventing disease in a subject        needing such treatment or prevention, the method comprising        administering the liposomal polyglutamated alpha        tetrahydrofolate composition of any of [12]-[69] to the subject;    -   [74] a method of killing a hyperproliferative cell that        comprises contacting a hyperproliferative cell with the        composition of any of [1]-[69];    -   [75] a method of killing a hyperproliferative cell that        comprises contacting a hyperproliferative cell with the        liposomal polyglutamated alpha tetrahydrofolate composition of        any of [12]-[69];    -   [76] the method of [74] or [75], wherein the hyperproliferative        cell is a cancer cell, a mammalian cell, and/or a human cell;    -   [77] a method for treating cancer that comprises administering        an effective amount of the composition of any of [1]-[69] to a        subject having or at risk of having cancer;    -   [78] a method for treating cancer that comprises administering        an effective amount of the liposomal polyglutamated alpha        tetrahydrofolate composition of any of [12]-[68] to a subject        having or at risk of having cancer;    -   [79] the method of [77] or [78], wherein the method treats or        prevents cancer and wherein the cancer is selected from the        group consisting of: a non-hematologic malignancy including such        as for example, lung cancer, pancreatic cancer, breast cancer,        ovarian cancer, prostate cancer, head and neck cancer, gastric        cancer, gastrointestinal cancer, colorectal cancer, esophageal        cancer, cervical cancer, liver cancer, kidney cancer, biliary        duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,        osteosarcoma), brain cancer, central nervous system cancer, and        melanoma; and a hematologic malignancy such as for example, a        leukemia, a lymphoma and other B cell malignancies, myeloma and        other plasma cell dyscrasias;    -   [80] the method of [77] or [78], wherein the method treats or        prevents cancer and wherein the cancer is a member selected from        the group consisting of: lung cancer, breast cancer, colon        cancer, pancreatic cancer, gastric cancer, bladder cancer, head        and neck cancer, ovarian cancer, and cervical cancer;    -   [81] the method of [77] or [78], wherein the method treats or        prevents cancer and wherein the cancer is a member selected from        the group consisting of: colorectal cancer, lung cancer, breast        cancer, head and neck cancer, and pancreatic cancer;    -   [82] the method of [77] or [78], wherein the method treats or        prevents cancer and wherein the cancer is selected from the        group consisting of: colorectal cancer, breast cancer, ovarian        cancer, lung cancer, head and neck cancer, pancreatic cancer,        gastric cancer, and mesothelioma;    -   [83] a method for treating cancer that comprises administering        an effective amount of the Lp-αPTHF composition of any of        [50]-[66] to a subject having or at risk of having a cancer cell        that expresses on its surface a folate receptor bound by the        targeting moiety;    -   [84] a maintenance therapy for subjects that are undergoing or        have undergone cancer therapy that comprise administering an        effective amount of the composition of any of [1]-[69] to a        subject that is undergoing or has undergone cancer therapy;    -   [85] a maintenance therapy for subjects that are undergoing or        have undergone cancer therapy that comprise administering an        effective amount of the liposomal polyglutamated alpha        tetrahydrofolate composition of any of [12]-[69] to a subject        that is undergoing or has undergone cancer therapy;    -   [86] a method for treating a disorder of the immune system that        comprises administering an effective amount of the composition        of any of [1]-[69] to a subject having or at risk of having a        disorder of the immune system, optionally wherein the disorder        of the immune system is selected from: inflammation (e.g., acute        and chronic), systemic inflammation, rheumatoid arthritis,        inflammatory bowel disease (IBD), Crohn disease,        dermatomyositis/polymyositis, systemic lupus erythematosus, and        Takayasu, and psoriasis;    -   [87] a method for treating a disorder of the immune system that        comprises administering an effective amount of the liposomal        polyglutamated alpha tetrahydrofolate composition of any of        [8]-[69] to a subject having or at risk of having a disorder of        the immune system, optionally wherein the disorder of the immune        system is selected from: inflammation (e.g., acute and chronic),        systemic inflammation, rheumatoid arthritis, inflammatory bowel        disease (IBD), Crohn disease, dermatomyositis/polymyositis,        systemic lupus erythematosus, and Takayasu, and psoriasis;    -   [88] a method for treating:        -   (a) leukopenia that comprises administering an effective            amount of the composition according to any of [1]-[69] to a            subject having or at risk of having leukopenia;        -   (b) an infectious disease that comprises administering an            effective amount of the composition according to any of            [1]-[69] to a subject having or at risk of having an            infectious disease;        -   (c) cardiovascular disease or metabolic disease that            comprises administering an effective amount of the            composition according to any of [1]-[69] to a subject having            or at risk of having an infectious disease, cardiovascular            disease, or another disease, wherein the disease is a member            selected from: atherosclerosis, cardiovascular disease            (CVD), coronary artery disease, myocardial infarction,            stroke, metabolic syndrome, a gestational trophoblastic            disease, and ectopic pregnancy;        -   (d) an autoimmune disease, that comprises administering an            effective amount of the composition according to any of            [1]-[69] to a subject having or at risk of having an            autoimmune disease;        -   (e) rheumatoid arthritis, that comprises administering an            effective amount of the composition according to any of            [1]-[69] to a subject having or at risk of having rheumatoid            arthritis;        -   (f) an inflammatory condition that comprises administering            an effective amount of the composition according to any of            [1]-[69] to a subject having or at risk of having            inflammation, optionally wherein the inflammation is acute,            chronic, and/or systemic inflammation; or    -   (g) a skin condition that comprises administering an effective        amount of the composition according to any of [1]-[69] to a        subject having or at risk of having a skin condition, optionally        wherein the skin condition is psoriasis;    -   [89] a method for treating an infectious disease that comprises        administering an effective amount of the liposomal        polyglutamated alpha tetrahydrofolate composition of any of        [12]-[69] to a subject having or at risk of having an infectious        disease;    -   [90] a method of delivering polyglutamated alpha        tetrahydrofolate to a tumor expressing a folate receptor on its        surface, the method comprising: administering the Lp-αPTHF        composition of any of [1]-[69] to a subject having the tumor in        an amount to deliver a therapeutically effective dose of the        polyglutamated alpha tetrahydrofolate to the tumor;    -   [91] a method of preparing a polyglutamated alpha        tetrahydrofolate composition comprising the liposomal        polyglutamated alpha tetrahydrofolate composition of any of        [12]-[69], the method comprising: forming a mixture comprising:        liposomal components and polyglutamated alpha antifolate in        solution; homogenizing the mixture to form liposomes in the        solution; and processing the mixture to form liposomes        containing polyglutamated alpha tetrahydrofolate;    -   [92] a method of preparing the composition of any of [12]-[69]        comprising the steps of: forming a mixture comprising: liposomal        components and polyglutamated alpha tetrahydrofolate in a        solution; homogenizing the mixture to form liposomes in the        solution; processing the mixture to form liposomes entrapping        and/or encapsulating polyglutamated alpha tetrahydrofolate; and        providing a targeting moiety on a surface of the liposomes, the        targeting moiety having specific affinity for at least one of        folate receptor alpha (FR-α), folate receptor beta (FR-β) and        folate receptor delta (FR-δ);    -   [93] the method according to [92], wherein the processing step        includes one or more steps of: thin film hydration, extrusion,        in-line mixing, ethanol injection technique,        freezing-and-thawing technique, reverse-phase evaporation,        dynamic high pressure microfluidization, microfluidic mixing,        double emulsion, freeze-dried double emulsion, 3D printing,        membrane contactor method, and stirring; and/or    -   [94] the method according to [92], wherein said processing step        includes one or more steps of modifying the size of the        liposomes by one or more of steps of extrusion, high-pressure        microfluidization, and/or sonication.

In some embodiments, the disclosure provides a polyglutamated alphatetrahydrofolate (αPTHF) composition wherein at least 2 of the glutamylresidues of the polyglutamated alpha tetrahydrofolate have a alphacarboxyl group linkage. In some embodiments, the αPTHF contains 2-20,2-15, 2-10, 2-5, or more than 5, glutamyl groups (including the glutamylgroup in tetrahydrofolate). In some embodiments, the αPTHF comprises twoor more glutamyl groups in the L-form. In other embodiments, the αPTHFcomprises a glutamyl group in the D-form. In further embodiments, theαPTHF comprises a glutamyl group in the D-form and two or more glutamylgroups in the L-form. In additional embodiments, the αPTHF comprises twoor more glutamyl groups that have an alpha linkage. In some embodiments,at least one glutamyl group has both an alpha linkage and a gammalinkage.

In one embodiment, the αPTHF composition contains a chain of 3 glutamylgroups attached to the glutamyl group of tetrahydrofolate (i.e., atetraglutamated tetrahydrofolate). In some embodiments, thetetraglutamated THF comprises two or more glutamyl groups in the L-form.In other embodiments, the tetraglutamated THF comprises a glutamyl groupin the D-form. In further embodiments, the tetraglutamated THF comprisesa glutamyl group in the D-form and two or more glutamyl groups in theL-form. In additional embodiments, the tetraglutamated THF comprises twoor more glutamyl groups that have a gamma linkage.

In one embodiment, the αPTHF composition contains a chain of 4 glutamylgroups attached to the gamma glutamyl group of tetrahydrofolate (e.g.,α-pentaglutamated tetrahydrofolate). In some embodiments, thepentaglutamated alpha THF comprises two or more glutamyl groups in theL-form. In other embodiments, the pentaglutamated alpha THF comprises aglutamyl group in the D-form. In further embodiments, thepentaglutamated alpha THF comprises a glutamyl group in the D-form andtwo or more glutamyl groups in the L-form. In additional embodiments,the pentaglutamated THF comprises two or more glutamyl groups that havea gamma linkage.

In one embodiment, the αPTHF composition contains a chain of 5 glutamylgroups attached to the gamma glutamyl group of tetrahydrofolate (e.g.,α-hexaglutamated tetrahydrofolate). In some embodiments, thehexaglutamated alpha THF comprises two or more glutamyl groups in theL-form. In other embodiments, the hexaglutamated alpha THF comprises aglutamyl group in the D-form. In further embodiments, the hexaglutamatedalpha THF comprises a glutamyl group in the D-form and two or moreglutamyl groups in the L-form. In additional embodiments, thehexaglutamated THF comprises two or more glutamyl groups that have agamma linkage.

In additional embodiments, the disclosure provides compositionscontaining delivery vehicles such as liposomes filled with (e.g.,encapsulating) and/or otherwise associated with polyglutamated alphatetrahydrofolate, and methods of making and using the αPTHFfilled/associated delivery vehicle compositions (DV-αPTHF) to deliverpolyglutamated alpha tetrahydrofolate to diseased (e.g., cancerous)and/or targeted cells. These compositions have uses that include but arenot limited to treating (e.g., treating or preventing) diseases thatinclude for example, hyperproliferative diseases such as cancer,disorders of the immune system such as inflammation and rheumatoidarthritis, and infectious disease such as HIV and malaria. In someembodiments, polyglutamated alpha tetrahydrofolate in the DV-αPTHFcontains 2-20, 2-15, 2-10, 2-5, more than 5, or more than 20, glutamylgroups (including the glutamyl group in tetrahydrofolate). The DV-αPTHFfilled/associated delivery vehicle compositions provide improvements tothe efficacy and safety of delivering tetrahydrofolate to cancer cellsby providing the preferential delivery of a more cytotoxic payload(e.g., polyglutamated tetrahydrofolate) compared to the cytotoxicity oftetrahydrofolate administered in its monoglutamate state (THF).

In some embodiments, the disclosure provides the use of compositionscontaining delivery vehicles such as liposomes filled with (e.g.,encapsulating) and/or otherwise associated with polyglutamated alphatetrahydrofolate in combination therapy with one or more therapeuticagents such as a chemotherapeutic drug (e.g., 5-fluorouracil) to enhancethe effectiveness of the therapeutic agent(s) or as a “chemoprotectant”(e.g., in combination with antifolates such as methotrexate) to reducetoxic side effects associated with the therapeutic agent(s). In someembodiments, polyglutamated alpha tetrahydrofolate in the DV-αPTHFcontains 2-20, 2-15, 2-10, 2-5, more than 5, or more than 20, glutamylgroups (including the glutamyl group in tetrahydrofolate). The DV-αPTHFfilled/associated delivery vehicle compositions provide improvements tothe efficacy and safety of delivering tetrahydrofolate to cancer cellsby providing the preferential delivery of a more cytotoxic payload(e.g., polyglutamated tetrahydrofolate) compared to the cytotoxicity oftetrahydrofolate administered in its monoglutamate state (THF).

In additional embodiments, the disclosure provides a compositioncomprising a polyglutamated alpha tetrahydrofolate (αPTHF).

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha 5-formyl-THF. In some embodiments, thepolyglutamated alpha 5-formyl-THF polyglutamated alpha[6S]-5-formyl-THF. In some embodiments, the composition comprisespolyglutamated alpha [6R,S]-5-formyl-THF. In some embodiments, thecomposition comprises polyglutamated alpha [6R]-5-formyl-THF. In someembodiments, the composition contains polyglutamated alpha 5-formyl-THFthat has 2-20, 2-15, 2-10, 2-5, or more than 20, glutamyl groups(including the glutamyl group in 5-formyl-THF). In some embodiments, thepolyglutamated alpha 5-formyl-THF comprises 1, 2, 3, or more than 3,glutamyl groups that have an alpha linkage. In some embodiments, thecomposition contains polyglutamated alpha 5-formyl-THF that has two ormore glutamyl groups in the L-form. In other embodiments, thecomposition contains polyglutamated alpha 5-formyl-THF that has aglutamyl group in the D-form. In further embodiments, the compositioncontains polyglutamated alpha 5-formyl-THF that has a glutamyl group inthe D-form and two or more glutamyl groups in the L-form. In additionalembodiments, the polyglutamated alpha tetrahydrofolate in the Lp-αPTHFcomprises two or more glutamyl groups that have an alpha linkage. Inadditional embodiments, the polyglutamated alpha tetrahydrofolate in theLp-αPTHF comprises one or more glutamyl groups that have both an alphalinkage and a gamma linkage. In some embodiments, the polyglutamatedalpha tetrahydrofolate in the Lp-αPTHF comprises 2-10 glutamyl groupsthat have both an alpha linkage and a gamma linkage, or any rangetherein between. In some embodiments, the polyglutamate chain of thepolyglutamated alpha 5-formyl-THF is linear. In some embodiments, thepolyglutamate chain of the polyglutamated alpha 5-formyl-THF isbranched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha 5-formyl-THF that contains a chain of 3 glutamylgroups attached to the glutamyl group of tetrahydrofolate (i.e.,tetraglutamated 5-formyl-THF). In some embodiments, the compositioncomprises tetraglutamated alpha [6S]-5-formyl-THF. In some embodiments,the composition comprises tetraglutamated alpha [6R,S]-5-formyl-THF. Insome embodiments, the composition comprises tetraglutamated alpha[6R]-5-formyl-THF. In some embodiments, the tetraglutamated alpha5-formyl-THF comprises 1, 2, or 3, glutamyl groups that have an alphalinkage. In some embodiments, the tetraglutamated alpha 5-formyl-THFcomprises two or more glutamyl groups in the L-form. In otherembodiments, the tetraglutamated alpha 5-formyl-THF comprises a glutamylgroup in the D-form. In further embodiments, the tetraglutamated alpha5-formyl-THF comprises a glutamyl group in the D-form and two or moreglutamyl groups in the L-form. In some embodiments, the polyglutamatechain of the tetraglutamated alpha 5-formyl-THF is linear. In someembodiments, the polyglutamate chain of the tetraglutamated alpha5-formyl-THF is branched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha 5-formyl-THF that contains a chain of 4 glutamylgroups attached to the glutamyl group of tetrahydrofolate (i.e.,pentaglutamated 5-formyl-THF). In some embodiments, the compositioncomprises pentaglutamated alpha [68]-5-formyl-THF. In some embodiments,the composition comprises pentaglutamated alpha [6R,S]-5-formyl-THF. Insome embodiments, the composition comprises pentaglutamated alpha[6R]-5-formyl-THF. In some embodiments, the pentaglutamated alpha5-formyl-THF comprises 1, 2, 3, or 4, glutamyl groups that have an alphalinkage. In some embodiments, the pentaglutamated 5-formyl-THF comprisestwo or more glutamyl groups in the L-form. In other embodiments, thepentaglutamated 5-formyl-THF comprises a glutamyl group in the D-form.In further embodiments, the pentaglutamated 5-formyl-THF comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form. In some embodiments, the polyglutamate chain of thepentaglutamated alpha 5-formyl-THF is linear. In some embodiments, thepolyglutamate chain of the pentaglutamated alpha 5-formyl-THF isbranched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha 5-formyl-THF that contains a chain of 5 glutamylgroups attached to the glutamyl group of tetrahydrofolate (i.e.,hexaglutamated 5-formyl-THF). In some embodiments, the compositioncomprises hexaglutamated alpha [6S]-5-formyl-THF. In some embodiments,the composition comprises hexaglutamated alpha [6R,S]-5-formyl-THF. Insome embodiments, the composition comprises hexaglutamated alpha[6R]-5-formyl-THF. In some embodiments, the hexaglutamated alpha5-formyl-THF comprises 1, 2, 3, 4, or 5, glutamyl groups that have analpha linkage. In some embodiments, the hexaglutamated alpha5-formyl-THF comprises two or more glutamyl groups in the L-form. Inother embodiments, the hexaglutamated THF comprises a glutamyl group inthe D-form. In further embodiments, the hexaglutamated alpha5-formyl-THF comprises a glutamyl group in the D-form and two or moreglutamyl groups in the L-form. In some embodiments, the polyglutamatechain of the hexaglutamated alpha 5-formyl-THF is linear. In someembodiments, the polyglutamate chain of the hexaglutamated alpha5-formyl-THF is branched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha 5,10-methenyl-THF. In some embodiments, thecomposition comprises polyglutamated alpha [6R]-5,10-methenyl-THF. Insome embodiments, the composition comprises polyglutamated alpha[6R,S]-5,10-methenyl-THF. In some embodiments, the composition comprisespolyglutamated alpha [6S]-5,10-methenyl-THF. In some embodiments, thecomposition contains polyglutamated alpha 5,10-methenyl-THF that has2-20, 2-15, 2-10, 2-5, or more than 20, glutamyl groups (including theglutamyl group in 5,10-methenyl-THF). In some embodiments, thepolyglutamated alpha 5,10-methenyl-THF comprises 1, 2, 3, or more than3, glutamyl groups that have an alpha linkage. In some embodiments, thecomposition contains polyglutamated alpha 5-formyl-THF that has two ormore glutamyl groups in the L-form. In other embodiments, thecomposition contains polyglutamated alpha 5,10-methenyl-THF that has aglutamyl group in the D-form. In further embodiments, the compositioncontains polyglutamated alpha 5,10-methenyl-THF that has a glutamylgroup in the D-form and two or more glutamyl groups in the L-form. Insome embodiments, the polyglutamate chain of the polyglutamated alpha5,10-methenyl-THF is linear. In some embodiments, the polyglutamatechain of the polyglutamated alpha 5,10-methenyl-THF is branched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha 5,10-methenyl-THF that contains a chain of 3glutamyl groups attached to the glutamyl group of tetrahydrofolate(i.e., tetraglutamated 5,10-methenyl-THF). In some embodiments, thecomposition comprises tetraglutamated alpha [6R]-5,10-methenyl-THF. Insome embodiments, the composition comprises tetraglutamated alpha[6R,S]-5,10-methenyl-THF. In some embodiments, the composition comprisestetraglutamated alpha [6S]-5,10-methenyl-THF. In some embodiments, thetetraglutamated alpha 5,10-methenyl-THF comprises 1, 2, or 3, glutamylgroups that have an alpha linkage. In some embodiments, thetetraglutamated alpha 5,10-methenyl-THF comprises two or more glutamylgroups in the L-form. In other embodiments, the tetraglutamated alpha5,10-methenyl-THF comprises a glutamyl group in the D-form. In furtherembodiments, the tetraglutamated alpha 5,10-methenyl-THF comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form. In some embodiments, the polyglutamate chain of the5,10-methenyl-THF tetrahydrofolate is linear. In some embodiments, thepolyglutamate chain of the tetraglutamated alpha 5,10-methenyl-THF isbranched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha 5,10-methenyl-THF that contains a chain of 4glutamyl groups attached to the glutamyl group of tetrahydrofolate(i.e., pentaglutamated 5,10-methenyl-THF). In some embodiments, thecomposition comprises pentaglutamated alpha [6R]-5,10-methenyl-THF. Insome embodiments, the composition comprises pentaglutamated alpha[6R,S]-5,10-methenyl-THF. In some embodiments, the composition comprisespentaglutamated alpha [6S]-5,10-methenyl-THF. In some embodiments, thepentaglutamated alpha 5,10-methenyl-THF comprises 1, 2, 3, or 4,glutamyl groups that have an alpha linkage. In some embodiments, thepentaglutamated alpha 5,10-methenyl-THF comprises two or more glutamylgroups in the L-form. In other embodiments, the pentaglutamated alpha5,10-methenyl-THF comprises a glutamyl group in the D-form. In furtherembodiments, the pentaglutamated alpha 5,10-methenyl-THF comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form. In some embodiments, the polyglutamate chain of thepentaglutamated alpha 5,10-methenyl-THF is linear. In some embodiments,the polyglutamate chain of the pentaglutamated alpha 5,10-methenyl-THFis branched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha 5,10-methenyl-THF that contains a chain of 5glutamyl groups attached to the glutamyl group of tetrahydrofolate(i.e., hexaglutamated 5,10-methenyl-THF). In some embodiments, thecomposition comprises hexaglutamated alpha [6R]-5,10-methenyl-THF. Insome embodiments, the composition comprises hexaglutamated alpha[6R,S]-5,10-methenyl-THF. In some embodiments, the composition compriseshexaglutamated alpha [6S]-5,10-methenyl-THF. In some embodiments, thehexaglutamated alpha 5,10-methenyl-THF comprises 1, 2, 3, 4, or 5,glutamyl groups that have an alpha linkage. In some embodiments, thehexaglutamated alpha 5,10-methenyl-THF comprises two or more glutamylgroups in the L-form. In other embodiments, the hexaglutamated alpha5,10-methenyl-THF comprises a glutamyl group in the D-form. In furtherembodiments, the hexaglutamated alpha 5,10-methenyl-THF comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form. In some embodiments, the polyglutamate chain of thehexaglutamated alpha 5,10-methenyl-THF is linear. In some embodiments,the polyglutamate chain of the hexaglutamated alpha 5,10-methenyl-THF isbranched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha 5-methyl-THF. In some embodiments, the compositioncomprises polyglutamated alpha [6S]-5-methyl-THF. In some embodiments,the composition comprises polyglutamated alpha [6R,S]-5-methyl-THF. Insome embodiments, the composition comprises polyglutamated alpha[6R]-5-methyl-THF. In some embodiments, the composition containspolyglutamated alpha 5-methyl-THF that has 2-20, 2-15, 2-10, 2-5, ormore than 20, glutamyl groups (including the glutamyl group in5-methyl-THF). In some embodiments, the polyglutamated alpha5-methyl-THF comprises 1, 2, 3, or more than 3, glutamyl groups thathave an alpha linkage. In some embodiments, the composition containspolyglutamated alpha 5-methyl-THF that has two or more glutamyl groupsin the L-form. In other embodiments, the composition containspolyglutamated alpha 5-methyl-THF that has a glutamyl group in theD-form. In further embodiments, the composition contains polyglutamatedalpha 5-methyl-THF that has a glutamyl group in the D-form and two ormore glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain of the polyglutamated alpha 5-methyl-THF is linear.In some embodiments, the polyglutamate chain of the polyglutamated alpha5-methyl-THF is branched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha 5-methyl-THF that contains a chain of 3 glutamylgroups attached to the glutamyl group of tetrahydrofolate (i.e.,tetraglutamated 5-methyl-THF). In some embodiments, the compositioncomprises tetraglutamated alpha [6S]-5-methyl-THF. In some embodiments,the composition comprises tetraglutamated alpha [6R,S]-5-methyl-THF. Insome embodiments, the composition comprises tetraglutamated alpha[6R]-5-methyl-THF. In some embodiments, the tetraglutamated alpha5-methyl-THF comprises 1, 2, or 3, glutamyl groups that have a gammalinkage In some embodiments, the tetraglutamated alpha 5-methyl-THFcomprises two or more glutamyl groups in the L-form. In otherembodiments, the tetraglutamated alpha 5-methyl-THF comprises a glutamylgroup in the D-form. In further embodiments, the tetraglutamated alpha5-methyl-THF comprises a glutamyl group in the D-form and two or moreglutamyl groups in the L-form. In some embodiments, the polyglutamatechain of the tetraglutamated alpha 5-methyl-THF is linear. In someembodiments, the polyglutamate chain of the tetraglutamated alpha5-methyl-THF is branched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha 5-methyl-THF that contains a chain of 4 glutamylgroups attached to the glutamyl group of tetrahydrofolate (i.e.,pentaglutamated 5-methyl-THF). In some embodiments, the compositioncomprises pentaglutamated alpha [6S]-5-methyl-THF. In some embodiments,the composition comprises pentaglutamated alpha [6R,S]-5-methyl-THF. Insome embodiments, the composition comprises pentaglutamated alpha[6R]-5-methyl-THF. In some embodiments, the pentaglutamated alpha5-methyl-THF comprises 1, 2, 3, or 4, glutamyl groups that have a gammalinkage. In some embodiments, the pentaglutamated alpha 5-methyl-THFcomprises two or more glutamyl groups in the L-form. In otherembodiments, the pentaglutamated alpha 5-methyl-THF comprises a glutamylgroup in the D-form. In further embodiments, the pentaglutamated alpha5-methyl-THF comprises a glutamyl group in the D-form and two or moreglutamyl groups in the L-form. In some embodiments, the polyglutamatechain of the pentaglutamated alpha 5-methyl-THF is linear. In someembodiments, the polyglutamate chain of the pentaglutamated alpha5-methyl-THF is branched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha 5-methyl-THF that that contains a chain of 5glutamyl groups attached to the glutamyl group of tetrahydrofolate(i.e., hexaglutamated 5-methyl-THF). In some embodiments, thecomposition comprises hexaglutamated alpha [6S]-5-methyl-THF. In someembodiments, the composition comprises hexaglutamated alpha[6R,S]-5-methyl-THF. In some embodiments, the composition compriseshexaglutamated alpha [6R]-5-methyl-THF. In some embodiments, thehexaglutamated alpha 5-methyl-THF comprises 1, 2, 3, 4, or 5, glutamylgroups that have an alpha linkage. In some embodiments, thehexaglutamated alpha 5-methyl-THF comprises two or more glutamyl groupsin the L-form. In other embodiments, the hexaglutamated alpha5-methyl-THF comprises a glutamyl group in the D-form. In furtherembodiments, the hexaglutamated alpha 5-methyl-THF comprises a glutamylgroup in the D-form and two or more glutamyl groups in the L-form. Insome embodiments, the polyglutamate chain of the hexaglutamated alpha5-methyl-THF is linear. In some embodiments, the polyglutamate chain ofthe hexaglutamated alpha 5-methyl-THF is branched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha Tetrahydrofolate THF. In some embodiments, thecomposition comprises polyglutamated alpha [6S]-Tetrahydrofolate THF. Insome embodiments, the composition comprises polyglutamated alpha[6R,S]-Tetrahydrofolate THF. In some embodiments, the compositioncomprises polyglutamated alpha [6R]-Tetrahydrofolate THF. In someembodiments, the composition contains polyglutamated alphaTetrahydrofolate THF that has 2-20, 2-15, 2-10, 2-5, or more than 20,glutamyl groups (including the glutamyl group in Tetrahydrofolate THF).In some embodiments, the alpha polyglutamated Tetrahydrofolate-THFcomprises 1, 2, 3, or more than 3, glutamyl groups that have an alphalinkage. In some embodiments, the composition contains polyglutamatedalpha Tetrahydrofolate THF that has two or more glutamyl groups in theL-form. In other embodiments, the composition contains polyglutamatedalpha Tetrahydrofolate THF that has a glutamyl group in the D-form. Infurther embodiments, the composition contains polyglutamated alphaTetrahydrofolate THF that has a glutamyl group in the D-form and two ormore glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain of the polyglutamated alpha Tetrahydrofolate THF islinear. In some embodiments, the polyglutamate chain of thepolyglutamated alpha Tetrahydrofolate THF is branched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha Tetrahydrofolate THF that contains a chain of 3glutamyl groups attached to the glutamyl group of Tetrahydrofolate THF(i.e., tetraglutamated Tetrahydrofolate THF). In some embodiments, thecomposition comprises tetraglutamated alpha [6S] Tetrahydrofolate THF.In some embodiments, the composition comprises tetraglutamated alpha[6R,S]-Tetrahydrofolate THF. In some embodiments, the compositioncomprises tetraglutamated alpha [6R]-Tetrahydrofolate THF. In someembodiments, the tetraglutamated alpha Tetrahydrofolate-THF comprises 1,2, or 3, glutamyl groups that have an alpha linkage. In someembodiments, the tetraglutamated alpha Tetrahydrofolate THF comprisestwo or more glutamyl groups in the L-form. In other embodiments, thetetraglutamated alpha Tetrahydrofolate THF comprises a glutamyl group inthe D-form. In further embodiments, the tetraglutamated alphaTetrahydrofolate THF comprises a glutamyl group in the D-form and two ormore glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain of the tetraglutamated alpha Tetrahydrofolate THF islinear. In some embodiments, the polyglutamate chain of thetetraglutamated alpha Tetrahydrofolate THF is branched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha Tetrahydrofolate THF that contains a chain of 4glutamyl groups attached to the glutamyl group of THF (i.e.,pentaglutamated Tetrahydrofolate THF). In some embodiments, thecomposition comprises pentaglutamated alpha [6S] Tetrahydrofolate THF.In some embodiments, the composition comprises pentaglutamated alpha[6R,S]-Tetrahydrofolate THF. In some embodiments, the compositioncomprises pentaglutamated alpha [6R]-Tetrahydrofolate THF. In someembodiments, the pentaglutamated alpha Tetrahydrofolate-THF comprises 1,2, 3, or 4, glutamyl groups that have an alpha linkage. In someembodiments, the pentaglutamated alpha THF comprises two or moreglutamyl groups in the L-form. In other embodiments, the pentaglutamatedalpha Tetrahydrofolate THF comprises a glutamyl group in the D-form. Infurther embodiments, the pentaglutamated alpha Tetrahydrofolate THFcomprises a glutamyl group in the D-form and two or more glutamyl groupsin the L-form. In some embodiments, the polyglutamate chain of thepentaglutamated alpha Tetrahydrofolate THF is linear. In someembodiments, the polyglutamate chain of the pentaglutamated alpha THF isbranched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha Tetrahydrofolate THF that contains a chain of 5glutamyl groups attached to the glutamyl group of Tetrahydrofolate THF(i.e., hexaglutamated Tetrahydrofolate THF). In some embodiments, thecomposition comprises hexaglutamated alpha [6S] Tetrahydrofolate THF. Insome embodiments, the composition comprises hexaglutamated alpha[6R,S]-Tetrahydrofolate THF. In some embodiments, the compositioncomprises hexaglutamated alpha [6R]-Tetrahydrofolate THF. In someembodiments, the hexaglutamated alpha Tetrahydrofolate-THF comprises 1,2, 3, 4, or 5, glutamyl groups that have an alpha linkage. In someembodiments, the hexaglutamated alpha Tetrahydrofolate TetrahydrofolateTHF comprises two or more glutamyl groups in the L-form. In otherembodiments, the hexaglutamated Tetrahydrofolate THF comprises aglutamyl group in the D-form. In further embodiments, the hexaglutamatedalpha Tetrahydrofolate THF comprises a glutamyl group in the D-form andtwo or more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain of the hexaglutamated alpha Tetrahydrofolate THF islinear. In some embodiments, the polyglutamate chain of thehexaglutamated alpha Tetrahydrofolate THF is branched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha 5,10-methylene-THF. In some embodiments, thecomposition comprises polyglutamated alpha [6R]-5,10-methylene-THF. Insome embodiments, the composition comprises polyglutamated alpha[6R,S]-5,10-methylene-THF. In some embodiments, the compositioncomprises polyglutamated alpha [6S]-5,10-methylene-THF. In someembodiments, the composition contains polyglutamated alpha5,10-methylene-THF that has 2-20, 2-15, 2-10, 2-5, or more than 20,glutamyl groups (including the glutamyl group in 5,10-methylene-THF). Insome embodiments, the polyglutamated alpha 5,10-methylene-THF comprises1, 2, 3, or more than 3, glutamyl groups that have an alpha linkage. Insome embodiments, the composition contains polyglutamated alpha5,10-methylene-THF that has two or more glutamyl groups in the L-form.In other embodiments, the composition contains polyglutamated alpha5,10-methylene-THF that has a glutamyl group in the D-form. In furtherembodiments, the composition contains polyglutamated alpha5,10-methylene-THF that has a glutamyl group in the D-form and two ormore glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain of the polyglutamated alpha 5,10-methylene-THF islinear. In some embodiments, the polyglutamate chain of thepolyglutamated alpha 5,10-methylene-THF is branched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha 5,10-methylene-THF that contains a chain of 3glutamyl groups attached to the glutamyl group of tetrahydrofolate(i.e., tetraglutamated 5,10-methylene-THF). In some embodiments, thecomposition comprises tetraglutamated alpha [6R]-5,10-methylene-THF. Insome embodiments, the composition comprises tetraglutamated alpha[6R,S]-5,10-methylene-THF. In some embodiments, the compositioncomprises tetraglutamated alpha [6S]-5,10-methylene-THF. In someembodiments, the tetraglutamated alpha 5,10-methylene-THF comprises 1,2, or 3 glutamyl groups that have an alpha linkage. In some embodiments,the tetraglutamated alpha 5,10-methylene-THF comprises two or moreglutamyl groups in the L-form. In other embodiments, the tetraglutamatedalpha 5,10-methylene-THF comprises a glutamyl group in the D-form. Infurther embodiments, the tetraglutamated alpha 5,10-methylene-THFcomprises a glutamyl group in the D-form and two or more glutamyl groupsin the L-form. In some embodiments, the polyglutamate chain of thetetraglutamated alpha 5,10-methylene-THF is linear. In some embodiments,the polyglutamate chain of the tetraglutamated alpha 5,10-methylene-THFis branched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha 5,10-methylene-THF that contains a chain of 4glutamyl groups attached to the glutamyl group of tetrahydrofolate(i.e., pentaglutamated 10-methylene-THF). In some embodiments, thecomposition comprises pentaglutamated alpha [6R] 5,10-methylene-THF. Insome embodiments, the composition comprises pentaglutamated alpha[6R,S]-5,10-methylene-THF. In some embodiments, the compositioncomprises pentaglutamated alpha [6S]-5,10-methylene-THF. In someembodiments, the pentaglutamated alpha 5,10-methylene-THF comprises 1,2, 3, or 4, glutamyl groups that have an alpha linkage. In someembodiments, the pentaglutamated alpha 5,10-methylene-THF comprises twoor more glutamyl groups in the L-form. In other embodiments, thepentaglutamated alpha 5,10-methylene-THF comprises a glutamyl group inthe D-form. In further embodiments, the pentaglutamated alpha5,10-methylene-THF comprises a glutamyl group in the D-form and two ormore glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain of the pentaglutamated alpha 5,10-methylene-THF islinear. In some embodiments, the polyglutamate chain of thepentaglutamated alpha 5,10-methylene-THF is branched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha 5,10-methylene-THF that contains a chain of 5glutamyl groups attached to the glutamyl group of tetrahydrofolate(i.e., hexaglutamated 5,10-methylene-THF). In some embodiments, thecomposition comprises hexaglutamated alpha [6R] 5,10-methylene-THF. Insome embodiments, the composition comprises hexaglutamated alpha [6R]5,10-methylene-THF. In some embodiments, the composition compriseshexaglutamated alpha [6R,S]-5,10-methylene-THF. In some embodiments, thecomposition comprises hexaglutamated alpha [6S]-5,10-methylene-THF. Insome embodiments, the alpha hexa glutamated 5,10-methylene-THF comprises1, 2, 3, 4, or 5, glutamyl groups that have an alpha linkage. In someembodiments, the hexaglutamated alpha 5,10-methylene-THF comprises twoor more glutamyl groups in the L-form. In other embodiments, thehexaglutamated alpha 5,10-methylene-THF comprises a glutamyl group inthe D-form. In further embodiments, the hexaglutamated alpha5,10-methylene-THF comprises a glutamyl group in the D-form and two ormore glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain of the hexaglutamated alpha 5,10-methylene-THF islinear. In some embodiments, the polyglutamate chain of thehexaglutamated alpha 5,10-methylene-THF is branched.

In some embodiments, the disclosure provides a composition comprising apolyglutamated alpha 5-formimino-THF. In some embodiments, thecomposition comprises polyglutamated alpha [6S]-5-formimino-THF. In someembodiments, the composition comprises polyglutamated alpha[6R,S]-5-formimino-THF. In some embodiments, the composition comprisespolyglutamated alpha [6R]-5-formimino-THF. In some embodiments, thecomposition contains polyglutamated alpha 5-formimino-THF that has 2-20,2-15, 2-10, 2-5, or more than 20, glutamyl groups (including theglutamyl group in 5-formimino-THF). In some embodiments, thepolyglutamated alpha 5-formimino-THF comprises 1, 2, 3, or more than 3,glutamyl groups that have an alpha linkage. In some embodiments, thecomposition contains polyglutamated alpha 5-formimino-THF that has twoor more glutamyl groups in the L-form. In other embodiments, thecomposition contains polyglutamated alpha 5-formimino-THF that has aglutamyl group in the D-form. In further embodiments, the compositioncontains polyglutamated alpha 5-formimino-THF that has a glutamyl groupin the D-form and two or more glutamyl groups in the L-form. In someembodiments, the polyglutamate chain of the polyglutamated alpha5-formimino-THF is linear. In some embodiments, the polyglutamate chainof the polyglutamated alpha 5-formimino-THF is branched.

In one embodiment, the composition comprises a polyglutamated alpha5-formimino-THF that contains a chain of 3 glutamyl groups attached tothe glutamyl group of tetrahydrofolate (i.e., tetraglutamated5-formimino-THF). In some embodiments, the composition comprisestetraglutamated alpha [6S]-5-formimino-THF. In some embodiments, thecomposition comprises tetraglutamated alpha [6R,S]-5-formimino-THF. Insome embodiments, the composition comprises tetraglutamated alpha [6R]5-formimino-THF. In some embodiments, the tetraglutamated alpha5-formimino-THF comprises 1, 2, or 3, glutamyl groups that have an alphalinkage. In some embodiments, the tetraglutamated alpha 5-formimino-THFcomprises two or more glutamyl groups in the L-form. In otherembodiments, the tetraglutamated alpha 5-formimino-THF comprises aglutamyl group in the D-form. In further embodiments, thetetraglutamated alpha 5-formimino-THF comprises a glutamyl group in theD-form and two or more glutamyl groups in the L-form. In someembodiments, the polyglutamate chain of the tetraglutamated alpha5-formimino-THF is linear. In some embodiments, the polyglutamate chainof the tetraglutamated alpha 5-formimino-THF is branched.

In one embodiment, the composition comprises a polyglutamated alpha5-formimino-THF that contains a chain of 4 glutamyl groups attached tothe glutamyl group of tetrahydrofolate (i.e., pentaglutamated5-formimino-THF). In some embodiments, the composition comprisespentaglutamated alpha [6S]-5-formimino-THF. In some embodiments, thecomposition comprises pentaglutamated alpha [6R,S]-5-formimino-THF. Insome embodiments, the composition comprises pentaglutamated alpha [6R]5-formimino-THF. In some embodiments, the pentaglutamated alpha5-formimino-THF comprises 1, 2, 3, or 4, glutamyl groups that have analpha linkage. In some embodiments, the pentaglutamated alpha5-formimino-THF comprises two or more glutamyl groups in the L-form. Inother embodiments, the pentaglutamated alpha 5-formimino-THF comprises aglutamyl group in the D-form. In further embodiments, thepentaglutamated alpha 5-formimino-THF comprises a glutamyl group in theD-form and two or more glutamyl groups in the L-form. In someembodiments, the polyglutamate chain of the pentaglutamated alpha5-formimino-THF is linear. In some embodiments, the polyglutamate chainof the pentaglutamated alpha 5-formimino-THF is branched.

In one embodiment, the composition comprises a polyglutamated alpha5-formimino-THF that contains a chain of 5 glutamyl groups attached tothe glutamyl group of tetrahydrofolate (i.e., hexaglutamated5-formimino-THF). In some embodiments, the composition compriseshexaglutamated alpha [6S]-5-formimino-THF. In some embodiments, thecomposition comprises hexaglutamated alpha [6R,S]-5-formimino-THF. Insome embodiments, the composition comprises hexaglutamated alpha [6R]5-formimino-THF. In some embodiments, the hexaglutamated alpha5-formimino-THF comprises 1, 2, 3, 4, or 5, glutamyl groups that have analpha linkage. In some embodiments, the hexaglutamated alpha5-formimino-THF comprises two or more glutamyl groups in the L-form. Inother embodiments, the hexaglutamated alpha 5-formimino-THF comprises aglutamyl group in the D-form. In further embodiments, the hexaglutamatedalpha 5-formimino-THF comprises a glutamyl group in the D-form and twoor more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain of the hexaglutamated alpha 5-formimino-THF islinear. In some embodiments, the polyglutamate chain of thehexaglutamated alpha 5-formimino-THF is branched

In additional embodiments, the disclosure provides a compositioncomprising a liposome encapsulating (filled with) polyglutamated alphatetrahydrofolate (Lp-αPTHF).

In some embodiments, the disclosure provides a composition comprising aliposome encapsulating (filled with) polyglutamated alpha 5-formyl-THF.In some embodiments, the liposome comprises polyglutamated alpha[6S]-5-formyl-THF. In some embodiments, the liposome comprisespolyglutamated alpha [6R,S]-5-formyl-THF. In some embodiments, theliposome comprises polyglutamated alpha [6R]-5-formyl-THF. In someembodiments, the liposome contains polyglutamated alpha 5-formyl-THFthat has 2-20, 2-15, 2-10, 2-5, or more than 20, glutamyl groups(including the glutamyl group in 5-formyl-THF). In some embodiments, thepolyglutamated alpha 5-formyl-THF comprises 1, 2, 3, or more than 3,glutamyl groups that have an alpha linkage. In some embodiments, theliposome contains polyglutamated alpha 5-formyl-THF that has two or moreglutamyl groups in the L-form. In other embodiments, the liposomecontains polyglutamated alpha 5-formyl-THF that has a glutamyl group inthe D-form. In further embodiments, the liposome contains polyglutamatedalpha 5-formyl-THF that has a glutamyl group in the D-form and two ormore glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain of the polyglutamated alpha 5-formyl-THF is linear.In some embodiments, the polyglutamate chain of the polyglutamated alpha5-formyl-THF is branched.

In one embodiment, the Lp-αPTHF composition comprises a polyglutamatedalpha 5-formyl-THF that contains a chain of 3 glutamyl groups attachedto the glutamyl group of tetrahydrofolate (i.e., tetraglutamated5-formyl-THF). In some embodiments, the liposome comprisestetraglutamated alpha [6S]-5-formyl-THF. In some embodiments, theliposome comprises tetraglutamated alpha [6R,S]-5-formyl-THF. In someembodiments, the liposome comprises tetraglutamated alpha[6R]-5-formyl-THF. In some embodiments, the tetraglutamated alpha5-formyl-THF comprises 1, 2, or 3, glutamyl groups that have a gammalinkage. In some embodiments, the tetraglutamated alpha 5-formyl-THFcomprises two or more glutamyl groups in the L-form. In otherembodiments, the tetraglutamated alpha 5-formyl-THF comprises a glutamylgroup in the D-form. In further embodiments, the tetraglutamated alpha5-formyl-THF comprises a glutamyl group in the D-form and two or moreglutamyl groups in the L-form. In some embodiments, the polyglutamatechain of the tetraglutamated alpha 5-formyl-THF is linear. In someembodiments, the polyglutamate chain of the tetraglutamated alpha5-formyl-THF is branched.

In one embodiment, the Lp-αPTHF composition comprises a polyglutamatedalpha 5-formyl-THF that contains a chain of 4 glutamyl groups attachedto the glutamyl group of tetrahydrofolate (i.e., pentaglutamated5-formyl-THF). In some embodiments, the liposome comprisespentaglutamated alpha [6S]-5-formyl-THF. In some embodiments, theliposome comprises pentaglutamated alpha [6R,S]-5-formyl-THF. In someembodiments, the liposome comprises pentaglutamated alpha[6R]-5-formyl-THF. In some embodiments, the pentaglutamated alpha5-formyl-THF comprises 1, 2, 3, or 4, glutamyl groups that have an alphalinkage. In some embodiments, the pentaglutamated 5-formyl-THF comprisestwo or more glutamyl groups in the L-form. In other embodiments, thepentaglutamated 5-formyl-THF comprises a glutamyl group in the D-form.In further embodiments, the pentaglutamated 5-formyl-THF comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form. In some embodiments, the polyglutamate chain of thepentaglutamated alpha 5-formyl-THF is linear. In some embodiments, thepolyglutamate chain of the pentaglutamated alpha 5-formyl-THF isbranched.

In one embodiment, the Lp-αPTHF composition comprises a polyglutamatedalpha 5-formyl-THF that contains a chain of 5 glutamyl groups attachedto the glutamyl group of tetrahydrofolate (i.e., hexaglutamated5-formyl-THF). In some embodiments, the liposome compriseshexaglutamated alpha [6S]-5-formyl-THF. In some embodiments, theliposome comprises hexaglutamated alpha [6R,S]-5-formyl-THF. In someembodiments, the liposome comprises hexaglutamated alpha[6R]-5-formyl-THF. In some embodiments, the hexaglutamated alpha5-formyl-THF comprises 1, 2, 3, 4, or 5, glutamyl groups that have analpha linkage. In some embodiments, the hexaglutamated alpha5-formyl-THF comprises two or more glutamyl groups in the L-form. Inother embodiments, the hexaglutamated THF comprises a glutamyl group inthe D-form. In further embodiments, the hexaglutamated alpha5-formyl-THF comprises a glutamyl group in the D-form and two or moreglutamyl groups in the L-form. In some embodiments, the polyglutamatechain of the hexaglutamated alpha 5-formyl-THF is linear. In someembodiments, the polyglutamate chain of the hexaglutamated alpha5-formyl-THF is branched.

In some embodiments, the disclosure provides a composition comprising aliposome encapsulating (filled with) polyglutamated alpha5,10-methenyl-THF. In some embodiments, the liposome comprisespolyglutamated alpha [6R]-5,10-methenyl-THF. In some embodiments, theliposome comprises polyglutamated alpha [6R,S]-5,10-methenyl-THF. Insome embodiments, the liposome comprises polyglutamated alpha[6S]-5,10-methenyl-THF. In some embodiments, the liposome containspolyglutamated alpha 5,10-methenyl-THF that has 2-20, 2-15, 2-10, 2-5,or more than 20, glutamyl groups (including the glutamyl group in5,10-methenyl-THF). In some embodiments, the polyglutamated alpha5,10-methenyl-THF comprises 1, 2, 3, or more than 3, glutamyl groupsthat have an alpha linkage. In some embodiments, the liposome containspolyglutamated alpha 5-formyl-THF that has two or more glutamyl groupsin the L-form. In other embodiments, the liposome containspolyglutamated alpha 5,10-methenyl-THF that has a glutamyl group in theD-form. In further embodiments, the liposome contains polyglutamatedalpha 5,10-methenyl-THF that has a glutamyl group in the D-form and twoor more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain of the polyglutamated alpha 5,10-methenyl-THF islinear. In some embodiments, the polyglutamate chain of thepolyglutamated alpha 5-formyl-THF is branched.

In one embodiment, the Lp-αPTHF composition comprises a polyglutamatedalpha 5,10-methenyl-THF that contains a chain of 3 glutamyl groupsattached to the glutamyl group of tetrahydrofolate (i.e.,tetraglutamated 5,10-methenyl-THF). In some embodiments, the liposomecomprises tetraglutamated alpha [6R]-5,10-methenyl-THF. In someembodiments, the liposome comprises tetraglutamated alpha[6R,S]-5,10-methenyl-THF. In some embodiments, the liposome comprisestetraglutamated alpha [6S]-5,10-methenyl-THF. In some embodiments, thetetraglutamated alpha 5,10-methenyl-THF comprises 1, 2, or 3, glutamylgroups that have an alpha linkage. In some embodiments, thetetraglutamated alpha 5,10-methenyl-THF comprises two or more glutamylgroups in the L-form. In other embodiments, the tetraglutamated alpha5,10-methenyl-THF comprises a glutamyl group in the D-form. In furtherembodiments, the tetraglutamated alpha 5,10-methenyl-THF comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form. In some embodiments, the polyglutamate chain of the5,10-methenyl-THF tetrahydrofolate is linear. In some embodiments, thepolyglutamate chain of the tetraglutamated alpha 5,10-methenyl-THF isbranched.

In one embodiment, the Lp-αPTHF composition comprises a polyglutamatedalpha 5,10-methenyl-THF that contains a chain of 4 glutamyl groupsattached to the glutamyl group of tetrahydrofolate (i.e.,pentaglutamated 5,10-methenyl-THF). In some embodiments, the liposomecomprises pentaglutamated alpha [6R]-5,10-methenyl-THF. In someembodiments, the liposome comprises pentaglutamated alpha[6R,S]-5,10-methenyl-THF. In some embodiments, the liposome comprisespentaglutamated alpha [6S]-5,10-methenyl-THF. In some embodiments, thepentaglutamated alpha 5,10-methenyl-THF comprises 1, 2, 3, or 4,glutamyl groups that have an alpha linkage. In some embodiments, thepentaglutamated alpha 5,10-methenyl-THF comprises two or more glutamylgroups in the L-form. In other embodiments, the pentaglutamated alpha5,10-methenyl-THF comprises a glutamyl group in the D-form. In furtherembodiments, the pentaglutamated alpha 5,10-methenyl-THF comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form. In some embodiments, the polyglutamate chain of thepentaglutamated alpha 5,10-methenyl-THF is linear. In some embodiments,the polyglutamate chain of the pentaglutamated alpha 5,10-methenyl-THFis branched.

In one embodiment, the Lp-αPTHF composition comprises a polyglutamatedalpha 5,10-methenyl-THF that contains a chain of 5 glutamyl groupsattached to the glutamyl group of tetrahydrofolate (i.e., hexaglutamated5,10-methenyl-THF). In some embodiments, the liposome compriseshexaglutamated alpha [6R]-5,10-methenyl-THF. In some embodiments, theliposome comprises hexaglutamated alpha [6R,S]-5,10-methenyl-THF. Insome embodiments, the liposome comprises hexaglutamated alpha[6S]-5,10-methenyl-THF. In some embodiments, the hexaglutamated alpha5,10-methenyl-THF comprises 1, 2, 3, 4, or 5, glutamyl groups that havean alpha linkage. In some embodiments, the hexaglutamated alpha5,10-methenyl-THF comprises two or more glutamyl groups in the L-form.In other embodiments, the hexaglutamated alpha 5,10-methenyl-THFcomprises a glutamyl group in the D-form. In further embodiments, thehexaglutamated alpha 5,10-methenyl-THF comprises a glutamyl group in theD-form and two or more glutamyl groups in the L-form. In someembodiments, the polyglutamate chain of the hexaglutamated alpha5,10-methenyl-THF is linear. In some embodiments, the polyglutamatechain of the hexaglutamated alpha 5,10-methenyl-THF is branched.

In some embodiments, the disclosure provides a composition comprising aliposome encapsulating (filled with) polyglutamated alpha 5-methyl-THF.In some embodiments, the liposome comprises polyglutamated alpha[6S]-5-methyl-THF. In some embodiments, the liposome comprisespolyglutamated alpha [6R,S]-5-methyl-THF. In some embodiments, theliposome comprises polyglutamated alpha [6R]-5-methyl-THF. In someembodiments, the liposome contains polyglutamated alpha 5-methyl-THFthat has 2-20, 2-15, 2-10, 2-5, or more than 20, glutamyl groups(including the glutamyl group in 5-methyl-THF). In some embodiments, thepolyglutamated alpha 5-methyl-THF comprises 1, 2, 3, or more than 3,glutamyl groups that have an alpha linkage. In some embodiments, theliposome contains polyglutamated alpha 5-methyl-THF that has two or moreglutamyl groups in the L-form. In other embodiments, the liposomecontains polyglutamated alpha 5-methyl-THF that has a glutamyl group inthe D-form. In further embodiments, the liposome contains polyglutamatedalpha 5-methyl-THF that has a glutamyl group in the D-form and two ormore glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain of the polyglutamated alpha 5-methyl-THF is linear.In some embodiments, the polyglutamate chain of the polyglutamated alpha5-methyl-THF is branched.

In one embodiment, the Lp-αPTHF composition comprises a polyglutamatedalpha 5-methyl-THF that contains a chain of 3 glutamyl groups attachedto the glutamyl group of tetrahydrofolate (i.e., tetraglutamated5-methyl-THF). In some embodiments, the liposome comprisestetraglutamated alpha [6S]-5-methyl-THF. In some embodiments, theliposome comprises tetraglutamated alpha [6R,S]-5-methyl-THF. In someembodiments, the liposome comprises tetraglutamated alpha[6R]-5-methyl-THF. In some embodiments, the tetraglutamated alpha5-methyl-THF comprises 1, 2, or 3, glutamyl groups that have an alphalinkage. In some embodiments, the tetraglutamated alpha 5-methyl-THFcomprises two or more glutamyl groups in the L-form. In otherembodiments, the tetraglutamated alpha 5-methyl-THF comprises a glutamylgroup in the D-form. In further embodiments, the tetraglutamated alpha5-methyl-THF comprises a glutamyl group in the D-form and two or moreglutamyl groups in the L-form. In some embodiments, the polyglutamatechain of the tetraglutamated alpha 5-methyl-THF is linear. In someembodiments, the polyglutamate chain of the tetraglutamated alpha5-methyl-THF is branched.

In one embodiment, the Lp-αPTHF composition comprises a polyglutamatedalpha 5-methyl-THF that contains a chain of 4 glutamyl groups attachedto the glutamyl group of tetrahydrofolate (i.e., pentaglutamated5-methyl-THF). In some embodiments, the liposome comprisespentaglutamated alpha [6S]-5-methyl-THF. In some embodiments, theliposome comprises pentaglutamated alpha [6R,S]-5-methyl-THF. In someembodiments, the liposome comprises pentaglutamated alpha[6R]-5-methyl-THF. In some embodiments, the pentaglutamated alpha5-methyl-THF comprises 1, 2, 3, or 4, glutamyl groups that have an alphalinkage. In some embodiments, the pentaglutamated alpha 5-methyl-THFcomprises two or more glutamyl groups in the L-form. In otherembodiments, the pentaglutamated alpha 5-methyl-THF comprises a glutamylgroup in the D-form. In further embodiments, the pentaglutamated alpha5-methyl-THF comprises a glutamyl group in the D-form and two or moreglutamyl groups in the L-form. In some embodiments, the polyglutamatechain of the pentaglutamated alpha 5-methyl-THF is linear. In someembodiments, the polyglutamate chain of the pentaglutamated alpha5-methyl-THF is branched.

In one embodiment, the Lp-αPTHF composition comprises a polyglutamatedalpha 5-methyl-THF that that contains a chain of 5 glutamyl groupsattached to the glutamyl group of tetrahydrofolate (i.e., hexaglutamated5-methyl-THF). In some embodiments, the liposome compriseshexaglutamated alpha [6S]-5-methyl-THF. In some embodiments, theliposome comprises hexaglutamated alpha [6R,S]-5-methyl-THF. In someembodiments, the liposome comprises hexaglutamated alpha[6R]-5-methyl-THF. In some embodiments, the hexaglutamated alpha5-methyl-THF comprises 1, 2, 3, 4, or 5, glutamyl groups that have analpha linkage. In some embodiments, the hexaglutamated alpha5-methyl-THF comprises two or more glutamyl groups in the L-form. Inother embodiments, the hexaglutamated alpha 5-methyl-THF comprises aglutamyl group in the D-form. In further embodiments, the hexaglutamatedalpha 5-methyl-THF comprises a glutamyl group in the D-form and two ormore glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain of the hexaglutamated alpha 5-methyl-THF is linear.In some embodiments, the polyglutamate chain of the hexaglutamated alpha5-methyl-THF is branched.

In some embodiments, the disclosure provides a composition comprising aliposome encapsulating (filled with) polyglutamated alphaTetrahydrofolate THF. In some embodiments, the liposome comprisespolyglutamated alpha [6S]-Tetrahydrofolate THF. In some embodiments, theliposome comprises polyglutamated alpha [6R,S]-Tetrahydrofolate THF. Insome embodiments, the liposome comprises polyglutamated alpha[6R]-Tetrahydrofolate THF. In some embodiments, the liposome containspolyglutamated alpha Tetrahydrofolate THF that has 2-20, 2-15, 2-10,2-5, or more than 20, glutamyl groups (including the glutamyl group inTetrahydrofolate THF). In some embodiments, the polyglutamated alphaTetrahydrofolate THF comprises 1, 2, 3, or more than 3, glutamyl groupsthat have an alpha linkage. In some embodiments, the liposome containspolyglutamated alpha Tetrahydrofolate THF that has two or more glutamylgroups in the L-form. In other embodiments, the liposome containspolyglutamated alpha Tetrahydrofolate THF that has a glutamyl group inthe D-form. In further embodiments, the liposome contains polyglutamatedalpha Tetrahydrofolate THF that has a glutamyl group in the D-form andtwo or more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain of the polyglutamated alpha Tetrahydrofolate THF islinear. In some embodiments, the polyglutamate chain of thepolyglutamated alpha Tetrahydrofolate THF is branched.

In one embodiment, the Lp-αPTetrahydrofolate THF composition comprises apolyglutamated alpha tetrahydrofolate that contains a chain of 3glutamyl groups attached to the glutamyl group of Tetrahydrofolate THF(i.e., tetraglutamated tetrahydrofolate). In some embodiments, theliposome comprises tetraglutamated alpha [6S] tetrahydrofolate. In someembodiments, the liposome comprises tetraglutamated alpha[6R,S]-Tetrahydrofolate THF. In some embodiments, the liposome comprisestetraglutamated alpha [6R]-Tetrahydrofolate THF. In some embodiments,the tetraglutamated alpha Tetrahydrofolate THF comprises 1, 2, or 3glutamyl groups that have an alpha linkage. In some embodiments, thetetraglutamated alpha Tetrahydrofolate THF comprises two or moreglutamyl groups in the L-form. In other embodiments, the tetraglutamatedalpha Tetrahydrofolate THF comprises a glutamyl group in the D-form. Infurther embodiments, the tetraglutamated alpha Tetrahydrofolate THFcomprises a glutamyl group in the D-form and two or more glutamyl groupsin the L-form. In some embodiments, the polyglutamate chain of thetetraglutamated alpha tetrahydrofolate is linear. In some embodiments,the polyglutamate chain of the tetraglutamated alpha tetrahydrofolate isbranched.

In one embodiment, the Lp-αPTetrahydrofolate THF composition comprises apolyglutamated alpha tetrahydrofolate that contains a chain of 4glutamyl groups attached to the glutamyl group of Tetrahydrofolate THF(i.e., pentaglutamated tetrahydrofolate). In some embodiments, theliposome comprises pentaglutamated alpha [6S] tetrahydrofolate. In someembodiments, the liposome comprises pentaglutamated alpha[6R,S]-Tetrahydrofolate THF. In some embodiments, the liposome comprisespentaglutamated alpha [6R]-Tetrahydrofolate THF. In some embodiments,the pentaglutamated alpha Tetrahydrofolate THF comprises 1, 2, 3, or 4,glutamyl groups that have an alpha linkage. In some embodiments, thepentaglutamated alpha Tetrahydrofolate THF comprises two or moreglutamyl groups in the L-form. In other embodiments, the pentaglutamatedalpha Tetrahydrofolate THF comprises a glutamyl group in the D-form. Infurther embodiments, the pentaglutamated alpha Tetrahydrofolate THFcomprises a glutamyl group in the D-form and two or more glutamyl groupsin the L-form. In some embodiments, the polyglutamate chain of thepentaglutamated alpha Tetrahydrofolate THF is linear. In someembodiments, the polyglutamate chain of the pentaglutamated alphaTetrahydrofolate THF is branched.

In one embodiment, the Lp-αPTetrahydrofolate THF composition comprises apolyglutamated alpha tetrahydrofolate that contains a chain of 5glutamyl groups attached to the glutamyl group of tetrahydrofolate(i.e., hexaglutamated tetrahydrofolate). In some embodiments, theliposome comprises hexaglutamated alpha [6S] tetrahydrofolate. In someembodiments, the liposome comprises hexaglutamated alpha[6R,S]-Tetrahydrofolate THF. In some embodiments, the liposome compriseshexaglutamated alpha [6R]-Tetrahydrofolate THF. In some embodiments, thehexaglutamated alpha Tetrahydrofolate THF comprises 1, 2, 3, 4, or 5glutamyl groups that have an alpha linkage. In some embodiments, thehexaglutamated alpha Tetrahydrofolate THF comprises two or more glutamylgroups in the L-form. In other embodiments, the hexaglutamatedTetrahydrofolate THF comprises a glutamyl group in the D-form. Infurther embodiments, the hexaglutamated alpha Tetrahydrofolate THFcomprises a glutamyl group in the D-form and two or more glutamyl groupsin the L-form. In some embodiments, the polyglutamate chain of thehexaglutamated alpha tetrahydrofolate is linear. In some embodiments,the polyglutamate chain of the hexaglutamated alpha tetrahydrofolate isbranched.

In some embodiments, the disclosure provides a composition comprising aliposome encapsulating (filled with) polyglutamated alpha5,10-methylene-THF. In some embodiments, the liposome comprisespolyglutamated alpha [6R]-5,10-methylene-THF. In some embodiments, theliposome comprises polyglutamated alpha [6R,S]-5,10-methylene-THF. Insome embodiments, the liposome comprises polyglutamated alpha[6S]-5,10-methylene-THF. In some embodiments, the liposome containspolyglutamated alpha 5,10-methylene-THF that has 2-20, 2-15, 2-10, 2-5,or more than 20, glutamyl groups (including the glutamyl group in5,10-methylene-THF). In some embodiments, the polyglutamated alpha5,10-methylene-THF comprises 1, 2, 3, or more than 3, glutamyl groupsthat have an alpha linkage. In some embodiments, the liposome containspolyglutamated alpha 5,10-methylene-THF that has two or more glutamylgroups in the L-form. In other embodiments, the liposome containspolyglutamated alpha 5,10-methylene-THF that has a glutamyl group in theD-form. In further embodiments, the liposome contains polyglutamatedalpha 5,10-methylene-THF that has a glutamyl group in the D-form and twoor more glutamyl. In some embodiments, the polyglutamate chain of thepolyglutamated alpha 5,10-methylene-THF is linear. In some embodiments,the polyglutamate chain of the polyglutamated alpha 5,10-methylene-THFis branched.

In one embodiment, the Lp-αPTHF composition comprises a polyglutamatedalpha 5,10-methylene-THF that contains a chain of 3 glutamyl groupsattached to the glutamyl group of tetrahydrofolate (i.e.,tetraglutamated 5,10-methylene-THF). In some embodiments, the liposomecomprises tetraglutamated alpha [6R]-5,10-methylene-THF. In someembodiments, the liposome comprises tetraglutamated alpha[6R,S]-5,10-methylene-THF. In some embodiments, the liposome comprisestetraglutamated alpha [6S]-5,10-methylene-THF. In some embodiments, thetetraglutamated alpha 5,10-methylene-THF comprises 1, 2, or 3, glutamylgroups that have an alpha linkage. In some embodiments, thetetraglutamated alpha 5,10-methylene-THF comprises two or more glutamylgroups in the L-form. In other embodiments, the tetraglutamated alpha5,10-methylene-THF comprises a glutamyl group in the D-form. In furtherembodiments, the tetraglutamated alpha 5,10-methylene-THF comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form. In some embodiments, the polyglutamate chain of thetetraglutamated alpha 5,10-methylene-THF is linear. In some embodiments,the polyglutamate chain of the tetraglutamated alpha 5,10-methylene-THFis branched.

In one embodiment, the Lp-αPTHF composition comprises a polyglutamatedalpha 5,10-methylene-THF that contains a chain of 4 glutamyl groupsattached to the glutamyl group of tetrahydrofolate (i.e.,pentaglutamated 10-methylene-THF). In some embodiments, the liposomecomprises pentaglutamated alpha [6R] 5,10-methylene-THF. In someembodiments, the liposome comprises pentaglutamated alpha[6R,S]-5,10-methylene-THF. In some embodiments, the liposome comprisespentaglutamated alpha [6S]-5,10-methylene-THF. In some embodiments, thepentaglutamated alpha 5,10-methylene-THF comprises 1, 2, 3, or 4,glutamyl groups that have an alpha linkage. In some embodiments, thepentaglutamated alpha 5,10-methylene-THF comprises two or more glutamylgroups in the L-form. In other embodiments, the pentaglutamated alpha5,10-methylene-THF comprises a glutamyl group in the D-form. In furtherembodiments, the pentaglutamated alpha 5,10-methylene-THF comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form. In some embodiments, the polyglutamate chain of thepentaglutamated alpha 5,10-methylene-THF is linear. In some embodiments,the polyglutamate chain of the pentaglutamated alpha 5,10-methylene-THFis branched.

In one embodiment, the Lp-αPTHF composition comprises a polyglutamatedalpha 5,10-methylene-THF that contains a chain of 5 glutamyl groupsattached to the glutamyl group of tetrahydrofolate (i.e., hexaglutamated5,10-methylene-THF). In some embodiments, the liposome compriseshexaglutamated alpha [6R] 5,10-methylene-THF. In some embodiments, theliposome comprises hexaglutamated alpha [6R] 5,10-methylene-THF. In someembodiments, the liposome comprises hexaglutamated alpha[6R,S]-5,10-methylene-THF. In some embodiments, the liposome compriseshexaglutamated alpha [6S]-5,10-methylene-THF. In some embodiments, thehexaglutamated alpha 5,10-methylene-THF comprises 1, 2, 3, 4, or 5,glutamyl groups that have an alpha linkage. In some embodiments, thehexaglutamated alpha 5,10-methylene-THF comprises two or more glutamylgroups in the L-form. In other embodiments, the hexaglutamated alpha5,10-methylene-THF comprises a glutamyl group in the D-form. In furtherembodiments, the hexaglutamated alpha 5,10-methylene-THF comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form. In some embodiments, the polyglutamate chain of thehexaglutamated alpha 5,10-methylene-THF is linear. In some embodiments,the polyglutamate chain of the hexaglutamated alpha 5,10-methylene-THFis branched.

In some embodiments, the disclosure provides a composition comprising aliposome encapsulating (filled with) polyglutamated alpha5-formimino-THF. In some embodiments, the liposome comprisespolyglutamated alpha [6S]-5-formimino-THF. In some embodiments, theliposome comprises polyglutamated alpha [6R,S]-5-formimino-THF. In someembodiments, the liposome comprises polyglutamated alpha[6R]-5-formimino-THF. In some embodiments, the liposome containspolyglutamated alpha 5-formimino-THF that has 2-20, 2-15, 2-10, 2-5, ormore than 20, glutamyl groups (including the glutamyl group in5-formimino-THF). In some embodiments, the polyglutamated alpha5-formimino-THF comprises 1, 2, 3, or more than 3, glutamyl groups thathave an alpha linkage. In some embodiments, the liposome containspolyglutamated alpha 5-formimino-THF that has two or more glutamylgroups in the L-form. In other embodiments, the liposome containspolyglutamated alpha 5-formimino-THF that has a glutamyl group in theD-form. In further embodiments, the liposome contains polyglutamatedalpha 5-formimino-THF that has a glutamyl group in the D-form and two ormore glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain of the polyglutamated alpha 5-formimino-THF islinear. In some embodiments, the polyglutamate chain of thepolyglutamated alpha 5-formimino-THF is branched.

In one embodiment, the Lp-αPTHF composition comprises a polyglutamatedalpha 5-formimino-THF that contains a chain of 3 glutamyl groupsattached to the glutamyl group of tetrahydrofolate (i.e.,tetraglutamated 5-formimino-THF). In some embodiments, the liposomecomprises tetraglutamated alpha [6S]-5-formimino-THF. In someembodiments, the liposome comprises tetraglutamated alpha[6R,S]-5-formimino-THF. In some embodiments, the liposome comprisestetraglutamated alpha [6R] 5-formimino-THF. In some embodiments, thetetraglutamated alpha 5-formimino-THF comprises 1, 2, or 3, glutamylgroups that have an alpha linkage. In some embodiments, thetetraglutamated alpha 5-formimino-THF comprises two or more glutamylgroups in the L-form. In other embodiments, the tetraglutamated alpha5-formimino-THF comprises a glutamyl group in the D-form. In furtherembodiments, the tetraglutamated alpha 5-formimino-THF comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form. In some embodiments, the polyglutamate chain of thetetraglutamated alpha 5-formimino-THF is linear. In some embodiments,the polyglutamate chain of the tetraglutamated alpha 5-formimino-THF isbranched.

In one embodiment, the Lp-αPTHF composition comprises a polyglutamatedalpha 5-formimino-THF that contains a chain of 4 glutamyl groupsattached to the glutamyl group of tetrahydrofolate (i.e.,pentaglutamated 5-formimino-THF). In some embodiments, the liposomecomprises pentaglutamated alpha [6S]-5-formimino-THF. In someembodiments, the liposome comprises pentaglutamated alpha[6R,S]-5-formimino-THF. In some embodiments, the liposome comprisespentaglutamated alpha [6R] 5-formimino-THF. In some embodiments, thepentaglutamated alpha 5-formimino-THF comprises 1, 2, 3, or 4, glutamylgroups that have an alpha linkage. In some embodiments, thepentaglutamated alpha 5-formimino-THF comprises two or more glutamylgroups in the L-form. In other embodiments, the pentaglutamated alpha5-formimino-THF comprises a glutamyl group in the D-form. In furtherembodiments, the pentaglutamated alpha 5-formimino-THF comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form. In some embodiments, the polyglutamate chain of thepentaglutamated alpha 5-formimino-THF is linear. In some embodiments,the polyglutamate chain of the pentaglutamated alpha 5-formimino-THF isbranched.

In one embodiment, the Lp-αPTHF composition comprises a polyglutamatedalpha-5-formimino-THF that contains a chain of 5 glutamyl groupsattached to the glutamyl group of tetrahydrofolate (i.e., hexaglutamated5-formimino-THF). In some embodiments, the liposome compriseshexaglutamated alpha [6S]-5-formimino-THF. In some embodiments, theliposome comprises hexaglutamated alpha [6R,S]-5-formimino-THF. In someembodiments, the liposome comprises hexaglutamated alpha [6R]5-formimino-THF. In some embodiments, the hexaglutamated alpha5-formimino-THF comprises 1, 2, 3, 4, or 5, glutamyl groups that have analpha linkage. In some embodiments, the hexaglutamated alpha5-formimino-THF comprises two or more glutamyl groups in the L-form. Inother embodiments, the hexaglutamated alpha 5-formimino-THF comprises aglutamyl group in the D-form. In further embodiments, the hexaglutamatedalpha 5-formimino-THF comprises a glutamyl group in the D-form and twoor more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain of the hexaglutamated alpha 5-formimino-THF islinear. In some embodiments, the polyglutamate chain of thehexaglutamated alpha 5-formimino-THF is branched

In some embodiments, the Lp-αPTHF composition is cationic. In someembodiments, the Lp-αPTHF liposome is cationic and has a diameter in therange of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to150 nm, or any range therein between. In further embodiments, theLp-αPTHF liposome is cationic and the composition has a diameter in therange of 80 nm to 120 nm, or any range therein between. In someembodiments, the cationic Lp-αPTHF composition comprises at least 1%,5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,or more than 75%, w/w of the polyglutamated alpha THF. In someembodiments, during the process of preparing the Lp-αPTHF, at least 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, or more than 75%, of the starting material of polyglutamated alphaTHF is encapsulated (entrapped) in the cationic Lp-αPTHF. In additionalembodiments, the polyglutamated alpha tetrahydrofolate encapsulated bythe liposome is in a HEPES buffered solution within the liposome.

In other embodiments, Lp-αPTHF composition is anionic or neutral. Insome embodiments, the Lp-αPTHF liposome is anionic or neutral and has adiameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175nm, or 50 nm to 150 nm, or any range therein between. In furtherembodiments, the Lp-αPTHF liposome is anionic or neutral and thecomposition has a diameter in the range of 80 nm to 120 nm, or any rangetherein between. In some embodiments, the Lp-αPTHF liposome is anionicand has a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30nm to 175 nm, or 50 nm to 150 nm, or any range therein between. Infurther embodiments, the Lp-αPTHF liposome is anionic and thecomposition has a diameter in the range of 80 nm to 120 nm, or any rangetherein between. In some embodiments, the Lp-αPTHF liposome is neutraland has a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30nm to 175 nm, or 50 nm to 150 nm, or any range therein between. In someembodiments, the anionic or neutral Lp-αPTHF composition comprises atleast 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, or more than 75%, w/w of the polyglutamated alpha THF. In someembodiments, during the process of preparing the Lp-αPTHF, at least 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, or more than 75%, of the starting material of polyglutamated alphaTHF is encapsulated (entrapped) in the anionic or neutral Lp-αPTHF. Insome embodiments, the anionic or neutral Lp-αPTHF composition comprisesat least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, or more than 75%, w/w of the tetraglutamated alpha THF.In some embodiments, the anionic or neutral Lp-αPTHF compositioncomprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the pentaglutamatedalpha THF. In some embodiments, the anionic or neutral Lp-αPTHFcomposition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of thehexaglutamated alpha THF. In additional embodiments, the polyglutamatedalpha tetrahydrofolate encapsulated by the liposome is in a HEPESbuffered solution within the liposome.

In additional embodiments, the liposomal polyglutamated alphatetrahydrofolate composition is pegylated (PLp-αPTHF).

In some embodiments, the liposomal polyglutamated alpha tetrahydrofolatecomposition is non-targeted (NTLp-αPTHF). That is, the NTLp-αPTHFcomposition does not have specific affinity towards an epitope (e.g., anepitope of a surface antigen) expressed on the surface of a target cellof interest. In further embodiments, the non-targeted liposomalpolyglutamated alpha tetrahydrofolate composition is pegylated(NTPLp-αPTHF).

In other embodiments, the liposomal polyglutamated alphatetrahydrofolate composition is targeted (TLp-αPTHF). That is, theTLp-αPTHF composition contains a targeting moiety that has specificaffinity for an epitope (surface antigen) on a target cell of interest.In some embodiments, the targeting moiety of the TLp-αPTHF or TPLp-αPTHFis not attached to the liposome through a covalent bond. In otherembodiments, the targeting moiety of the TLp-αPTHF or TPLp-αPTHF isattached to one or both of a PEG and the exterior of the liposome.Targeted liposomal polyglutamated alpha tetrahydrofolate compositions(TLp-αPTHF and TPLp-αPTHF) provide further improvements over theefficacy and safety profile of tetrahydrofolate, by specificallydelivering polyglutamated alpha (e.g., α-pentaglutamated and/orα-hexaglutamated) tetrahydrofolate to target cells such as cancer cells.In some embodiments, the targeted liposomal polyglutamated alphatetrahydrofolate composition is pegylated (TPLp-αPTHF). In someembodiments, the targeting moiety of the TLp-αPTHF or TPLp-αPTHF isattached to one or both of a PEG and the exterior of the liposome. Insome embodiments, the targeting moiety of the TLp-αPTHF or TPLp-αPTHF isattached to the liposome through a covalent bond. Function of thetargeting moiety of the TLp-αPTHF and/or TPLp-αPTHF compositions includebut are not limited to, targeting the liposome to the target cell ofinterest in vivo or in vitro; interacting with the surface antigen forwhich the targeting moiety has specific affinity, and delivering theliposome payload (αPTHF) into the cell. Suitable targeting moieties areknown in the art and include, but are not limited to, antibodies,antigen-binding antibody fragments, scaffold proteins, polypeptides, andpeptides. In some embodiments, the targeting moiety is a polypeptide. Infurther embodiments, the targeting moiety is a polypeptide thatcomprises at least 3, 5, 10, 15, 20, 30, 40, 50, or 100, amino acidresidues.

In some embodiments, the targeting moiety of the TLp-αPTHF or TPLp-αPTHFis an antibody or an antigen-binding antibody fragment. In furtherembodiments, the targeting moiety comprises one or more of an antibody,a humanized antibody, an antigen binding fragment of an antibody, asingle chain antibody, a single-domain antibody, a bi-specific antibody,a synthetic antibody, a pegylated antibody, and a multimeric antibody.In some embodiments, the targeting moiety of the TLp-αPTHF or TPLp-αPTHFhas specific affinity for an epitope that is preferentially expressed ona target cell such as a tumor cell, compared to normal or non-tumorcells. In some embodiments, the targeting moiety has specific affinityfor an epitope on a tumor cell surface antigen that is present on atumor cell but absent or inaccessible on a non-tumor cell. In someembodiments, the targeting moiety binds an epitope of interest with anequilibrium dissociation constant (Kd) in a range of 0.5×10⁻¹⁰ to10×10⁻⁶ as determined using BIACORE® analysis.

In particular embodiments, the TLp-αPTHF or TPLp-αPTHF targeting moietycomprises a polypeptide that specifically binds a folate receptor. Insome embodiments, the targeting moiety is an antibody or anantigen-binding antibody fragment. In some embodiments, the folatereceptor bound by the targeting moiety is one or more folate receptorsselected from the group consisting of: folate receptor alpha (FR-α,FOLR1), folate receptor beta (FR-β, FOLR2), and folate receptor delta(FR-δ, FOLR4). In some embodiments, the folate receptor bound by thetargeting moiety is folate receptor alpha (FR-α). In some embodiments,the folate receptor bound by the targeting moiety is folate receptorbeta (FR-β). In some embodiments, the targeting moiety specificallybinds FR-α and FR-β.

In additional embodiments, the Lp-αPTHF composition comprises one ormore of an immunostimulatory agent, a detectable marker, and amaleimide, disposed on at least one of the PEG and the exterior of theliposome. In some embodiments, the liposome αPTHF composition (e.g.,Lp-αPTHF, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF)is cationic. In other embodiments, the liposome αPTHF composition (e.g.,Lp-αPTHF, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF or TPLp-αPTHF)is anionic or neutral. In additional embodiments, the liposome of theliposome αPTHF composition (e.g., Lp-αPTHF, PLp-αPTHF, NTLp-αPTHF,NTPLp-αPTHF, TLp-αPTHF or TPLp-αPTHF) has a diameter in the range of 20nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, 50 nm to 150 nm, or anyrange therein between. In some embodiments, the liposome of theliposome-αPTHF composition has a diameter in the range of 30 nm to 175nm or 50 nm to 150 nm, or any range therein between. In furtherembodiments, the liposome of the liposome αPTHF composition has adiameter in the range of 80 nm to 120 nm, or any range therein between.In some embodiments, the liposome αPTHF composition is pegylated (e.g.,PLp-αPTHF, NTPLp-αPTHF, or TPLp-αPTHF). In some embodiments, theliposome αPTHF composition comprises a targeting moiety (e.g., TLp-αPTHFor TPLp-αPTHF). In further embodiments, the liposome αPTHF compositionis pegylated and targeted (e.g., TPLp-αPTHF). In some embodiments, theliposome αPTHF composition comprises polyglutamated alphatetrahydrofolate that contains 4, 5, 2-10, 4-6, or more than 5, glutamylgroups. In some embodiments, the liposome αPTHF composition comprisestetraglutamated alpha tetrahydrofolate. In some embodiments, theliposome αPTHF composition comprises pentaglutamated alphatetrahydrofolate. In other embodiments, the liposome αPTHF compositioncomprises hexaglutamated alpha tetrahydrofolate.

In some embodiments, the liposome compositions comprise a polyglutamatedalpha tetrahydrofolate that contains 4, 5, 2-10, 4-6, or more than 5,glutamyl groups and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of thepolyglutamated alpha THF. In some embodiments, the Lp-αPTHF compositioncomprises polyglutamated alpha tetrahydrofolate that contains 4, 5,2-10, 4-6, or more than 5, glutamyl groups and 1%-98.5% w/w of thepolyglutamated alpha THF. In some embodiments, the liposomes comprisepolyglutamated alpha tetrahydrofolate that contains 4, 5, 2-10, 4-6, ormore than 5, glutamyl groups and wherein during the process of preparingthe Lp-αPTHF, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting materialof polyglutamated alpha THF is encapsulated (entrapped) in the Lp-αPTHF.

In some embodiments, the liposome compositions comprise atetraglutamated alpha tetrahydrofolate and at least 1%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than75%, w/w of the tetraglutamated alpha THF. In some embodiments, theLp-αPTHF composition comprises tetraglutamated alpha tetrahydrofolateand 1%-98.5% w/w of the tetraglutamated alpha THF. In some embodiments,the liposomes comprise tetraglutamated alpha tetrahydrofolate andwherein during the process of preparing the Lp-αPTHF, at least 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, ormore than 75% of the starting material of tetraglutamated alpha THF isencapsulated (entrapped) in the Lp-αPTHF.

In some embodiments, the liposome compositions comprise apentaglutamated alpha tetrahydrofolate and at least 1%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than75%, w/w of the pentaglutamated alpha THF. In some embodiments, theLp-αPTHF composition comprises pentaglutamated alpha tetrahydrofolateand 1%-98.5% w/w of the pentaglutamated alpha THF. In some embodiments,the liposomes comprise pentaglutamated alpha tetrahydrofolate andwherein during the process of preparing the Lp-αPTHF, at least 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, ormore than 75% of the starting material of pentaglutamated alpha THF isencapsulated (entrapped) in the Lp-αPTHF.

In some embodiments, the liposome compositions comprise a hexaglutamatedalpha tetrahydrofolate and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w ofthe hexaglutamated alpha THF. In some embodiments, the Lp-αPTHFcomposition comprises hexaglutamated alpha tetrahydrofolate and 1%-98.5%w/w of the hexaglutamated alpha THF. In some embodiments, the liposomescomprise hexaglutamated alpha tetrahydrofolate and wherein during theprocess of preparing the Lp-αPTHF, at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% ofthe starting material of pentaglutamated alpha THF is encapsulated(entrapped) in the Lp-αPTHF.

Liposomal compositions comprising liposomes encapsulating αPTHF are alsoprovided. In some embodiments, the liposomal composition comprises apegylated αPTHF composition. In some embodiments, the liposomalcomposition comprises a αPTHF composition that is linked to or otherwiseassociated with a targeting moiety. In further embodiments, theliposomal composition comprises a αPTHF composition that is pegylatedand linked to or otherwise associated with a targeting moiety. In someembodiments, the liposomal composition comprises αPTHF that contains 4,5, 2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, theliposomal composition comprises tetraglutamated alpha tetrahydrofolate.In some embodiments, the liposomal composition comprises pentaglutamatedalpha tetrahydrofolate. In other embodiments, the liposomal compositioncomprises hexaglutamated alpha tetrahydrofolate.

In some embodiments, the liposomal composition comprises a liposomeαPTHF (e.g., Lp-αPTHF, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF,and TPLp-αPTHF). In some embodiments, the liposome αPTHF is pegylated(e.g., NTPLp-αPTHF, and TPLp-αPTHF). In some embodiments, the liposomeαPTHF comprises a targeting moiety that has a specific affinity for anepitope of an antigen on the surface of a target cell of interest suchas a cancer cell (e.g., TLp-αPTHF or TPLp-αPTHF)). In furtherembodiments, the liposomal composition comprises a liposome αPTHF thatis pegylated and further comprises a targeting moiety that has aspecific affinity for an epitope of an antigen on the surface of atarget cell of interest such as a cancer cell (e.g., TPLp-αPTHF). Insome embodiments, the liposomal composition comprises a liposome αPTHFthat is cationic. In other embodiments, the liposomal compositioncomprises a liposome αPTHF that is anionic or neutral. In additionalembodiments, the liposomal composition comprises a liposome αPTHF thathas a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, or anyrange therein between. In further embodiments, the liposome αPTHF has adiameter in the range of 80 nm to 120 nm, or any range therein between.

Pharmaceutical compositions comprising polyglutamated alphatetrahydrofolate (αPTHF) including delivery vehicles such as liposomeαPTHF are also provided. In some embodiments, the pharmaceuticalcomposition comprises a pegylated αPTHF composition. In someembodiments, the pharmaceutical composition comprise a αPTHF compositionthat is linked to or otherwise associated with a targeting moiety. Infurther embodiments, the pharmaceutical composition comprise a αPTHFcomposition that is pegylated and linked to or otherwise associated witha targeting moiety. In some embodiments, the pharmaceutical compositioncomprises αPTHF that contains 4, 5, 2-10, 4-6, or more than 5, glutamylgroups. In some embodiments, the pharmaceutical composition comprisestetraglutamated alpha tetrahydrofolate. In some embodiments, thepharmaceutical composition comprises pentaglutamated alphatetrahydrofolate. In other embodiments, the pharmaceutical compositioncomprises hexaglutamated alpha tetrahydrofolate.

In some embodiments, the pharmaceutical compositions comprise a liposomeαPTHF (e.g., Lp-αPTHF, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF,and TPLp-αPTHF). In some embodiments, the liposome αPTHF composition ispegylated (e.g., NTPLp-αPTHF, and TPLp-αPTHF). In some embodiments, theliposome αPTHF comprises a targeting moiety that has a specific affinityfor an epitope of an antigen on the surface of a target cell of interestsuch as a cancer cell (e.g., TLp-αPTHF or TPLp-αPTHF)). In furtherembodiments, the pharmaceutical composition comprises a liposome αPTHFcomposition that is pegylated and further comprises a targeting moietythat has a specific affinity for an epitope of an antigen on the surfaceof a target cell of interest such as a cancer cell (e.g., TPLp-αPTHF).In some embodiments, the pharmaceutical composition comprises a liposomeαPTHF that is cationic. In other embodiments, the pharmaceuticalcomposition comprises a liposome αPTHF that is anionic or neutral. Inadditional embodiments, the pharmaceutical composition comprises aliposome αPTHF that has a diameter in the range of 20 nm to 500 nm or 20nm to 500 nm, or any range therein between. In further embodiments, theliposome αPTHF composition has a diameter in the range of 80 nm to 120nm, or any range therein between.

In additional embodiments, the disclosure provides a method ofmodulating the activation, chemokine production, or metabolic activityof a cell that comprises contacting the cell with a compositioncomprising a polyglutamated alpha tetrahydrofolate (αPTHF) composition.In some embodiments, the contacted cell is a mammalian cell. In furtherembodiments, the contacted cell is a human cell. In some embodiments,the contacted cell is a hyperproliferative cell. In further embodiments,the cell is an immune cell. In some embodiments, the method is performedin vivo. In other embodiments, the method is performed in vitro. In someembodiments, the αPTHF contains 4, 5, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, the αPTHF composition comprisestetraglutamated alpha tetrahydrofolate. In some embodiments, the αPTHFcomposition comprises pentaglutamated alpha tetrahydrofolate. In otherembodiments, the αPTHF composition comprises hexaglutamated alphatetrahydrofolate.

In additional embodiments, the disclosure provides a method ofmodulating the activation, chemokine production, or metabolic activityof a cell that comprises contacting the cell with a liposome comprisinga polyglutamated alpha tetrahydrofolate (αPTHF) composition. In someembodiments, the contacted cell is a mammalian cell. In furtherembodiments, the contacted cell is a human cell. In some embodiments,the contacted cell is a hyperproliferative cell. In further embodiments,the cell is an immune cell. In some embodiments, the method is performedin vivo. In other embodiments, the method is performed in vitro. In someembodiments, the αPTHF contains 4, 5, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, the αPTHF composition comprisestetraglutamated alpha tetrahydrofolate. In some embodiments, the αPTHFcomposition comprises pentaglutamated alpha tetrahydrofolate. In otherembodiments, the αPTHF composition comprises hexaglutamated alphatetrahydrofolate.

In additional embodiments, the disclosure provides a method of killing acell that comprises contacting the cell with a composition comprising apolyglutamated alpha tetrahydrofolate (αPTHF) composition (e.g., a αPTHFherein). In some embodiments, the contacted cell is a mammalian cell. Infurther embodiments, the contacted cell is a human cell. In someembodiments, the contacted cell is a hyperproliferative cell. In furtherembodiments, the hyperproliferative cell is a cancer cell. In furtherembodiments, the contacted cancer cell is a primary cell or a cell froma cell line obtained/derived from a cancer selected from the groupconsisting of: a non-hematologic malignancy including such as forexample, lung cancer, pancreatic cancer, breast cancer, ovarian cancer,prostate cancer, head and neck cancer, gastric cancer, gastrointestinalcancer, colorectal cancer, esophageal cancer, cervical cancer, livercancer, kidney cancer, biliary duct cancer, gallbladder cancer, bladdercancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervoussystem cancer, and melanoma; and a hematologic malignancy such as forexample, a leukemia, a lymphoma and other B cell malignancies, myelomaand other plasma cell dysplasias or dyscrasias. In further embodiments,the contacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from a cancer selected from the group consisting of:breast cancer, head and neck cancer, lung cancer, stomach cancer,osteosarcoma, Non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia(ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma,and chorioadenoma, nonleukemic meningeal cancer, soft tissue sarcoma(desmoid tumors, aggressive fibromatosis, bladder cancer, and centralNervous System (CNS) lymphoma. In yet further embodiments, the cancercell is a primary cell or a cell from a cell line obtained/derived froma cancer selected from colorectal cancer, breast cancer, gastric cancer(e.g., stomach cancer), pancreatic cancer, liver cancer, lung cancer(e.g., non-small cell lung cancer and/or adenocarcinoma), head and neckcancer, ovarian cancer, gallbladder cancer, and basal cell cancer. Inparticular embodiments, the cancer cell is a primary cell or a cell froma cell line obtained/derived from colorectal cancer. In someembodiments, the αPTHF contains 4, 5, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, the αPTHF contains 4 glutamylgroups. In some embodiments, the αPTHF contains 5 glutamyl groups. Insome embodiments, the αPTHF contains 6 glutamyl groups. In someembodiments, the method is performed in vivo. In other embodiments, themethod is performed in vitro.

In additional embodiments, the disclosure provides a method of killing acell that comprises contacting the cell with a liposome containingpolyglutamated alpha tetrahydrofolate (e.g., an Lp-αPTHF such as,PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF or TPLp-αPTHF). In yetfurther embodiments, the contacted hyperproliferative cell is a cancercell. In further embodiments, the cancer cell is a primary cell or acell from a cell line obtained/derived from a cancer selected from thegroup consisting of: a non-hematologic malignancy including such as forexample, lung cancer, pancreatic cancer, breast cancer, ovarian cancer,prostate cancer, head and neck cancer, gastric cancer, gastrointestinalcancer, colorectal cancer, esophageal cancer, cervical cancer, livercancer, kidney cancer, biliary duct cancer, gallbladder cancer, bladdercancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervoussystem cancer, and melanoma; and a hematologic malignancy such as forexample, a leukemia, a lymphoma and other B cell malignancies, myelomaand other plasma cell dysplasias or dyscrasias. In further embodiments,the contacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from a cancer selected from the group consisting of:breast cancer, head and neck cancer, lung cancer, stomach cancer,osteosarcoma, Non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia(ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma,and chorioadenoma, nonleukemic meningeal cancer, soft tissue sarcoma(desmoid tumors, aggressive fibromatosis, bladder cancer, and centralNervous System (CNS) lymphoma. In some embodiments, the cancer cell is aprimary cell or a cell from a cell line obtained/derived from a cancerselected from colorectal cancer, breast cancer, gastric cancer (e.g.,stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g.,non-small cell lung cancer and/or adenocarcinoma), head and neck cancer,ovarian cancer, gallbladder cancer, and basal cell cancer. In particularembodiments, the cancer cell is a primary cell or a cell from a cellline obtained/derived from colorectal cancer. In some embodiments, themethod is performed in vivo. In other embodiments, the method isperformed in vitro. In some embodiments, the liposome contains a αPTHFcontaining 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the liposome comprises a αPTHF containing 4, 5, 2-10, 4-6,or more than 5, glutamyl groups. In some embodiments, the liposomecomprises a αPTHF containing 4 glutamyl groups. In some embodiments, theliposome comprises a αPTHF containing 5 glutamyl groups. In someembodiments, the liposome comprises a αPTHF containing 6 glutamylgroups. In some embodiments, the αPTHF contains 1, 2, 3, or more than 3,glutamyl groups having a gamma linkage.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a deliveryvehicle (e.g., an immunoconjugate or liposome) comprising polyglutamatedalpha tetrahydrofolate to a subject having or at risk of having cancer.In some embodiments, the delivery vehicle is an antibody-containingimmunoconjugate (comprising. e.g., a full-length IgG antibody, abispecific antibody, or a scFv). In some embodiments, the deliveryvehicle is a liposome (e.g., an Lp-αPTHF such as, PLp-αPTHF, NTLp-αPTHF,NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF). In some embodiments, theadministered delivery vehicle is pegylated. In some embodiments, theadministered delivery vehicle is not pegylated. In additionalembodiments, the administered delivery vehicle comprises a targetingmoiety that has a specific affinity for an epitope of an antigen on thesurface of a cancer cell. In additional embodiments, the deliveryvehicle comprises a targeting moiety that has specific affinity for anepitope of a cell surface antigen selected from the group consisting of:GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin,fibronectin extra-domain B (ED-B), VEGFR2 (CD309), tenascin, collagenIV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, the deliveryvehicle comprises a targeting moiety that specifically binds a cellsurface antigen(s) derived from, or determined to be expressed on, aspecific subject's cancer (tumor) such as a neoantigen. In someembodiments, the targeting moiety has specific affinity for an epitopeof a cell surface antigen(s) derived from or determined to be expressedon a specific subject's tumor such as a neoantigen. In some embodiments,the targeting moiety is an antibody or an antigen binding antibodyfragment. In some embodiments, the administered delivery vehiclecomprises αPTHF containing 4, 5, 2-10, 4-6, or more than 5, -glutamylgroups. In some embodiments, the administered delivery vehicle comprisesαPTHF containing 4 glutamyl groups. In some embodiments, theadministered delivery vehicle comprises αPTHF containing 5 glutamylgroups. In some embodiments, the administered delivery vehicle comprisesαPTHF containing 6 glutamyl groups. In some embodiments, the αPTHF is amember selected from: (a) polyglutamated 5-formyl-THF (e.g.,polyglutamated [6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF(e.g., polyglutamated [6R]-10-formyl-THF); (c) polyglutamated5,10-methenyl-THF (e.g., polyglutamated [6R]-5,10-methenyl-THF); (d)polyglutamated 5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF);(e) polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, the αPTHF is polyglutamated 5,10-methylene-THF. In furtherembodiments, the αPTHF is polyglutamated [6R]-5,10-methylene-THF. Inother embodiments, the αPTHF is polyglutamated[6R,S]-5,10-methylene-THF. In some embodiments, the αPTHF ispolyglutamated 5-methyl-THF. In further embodiments, the αPTHF is[6S]-5-methyl-THF. In other embodiments, the αPTHF is[6R,S]-5-methyl-THF. In some embodiments, the αPTHF is polyglutamated5-formyl-THF. In further embodiments, the αPTHF is polyglutamated[6S]-5-formyl-THF. In other embodiments, the αPTHF is polyglutamated[6R,S]-5-formyl-THF. In some embodiments, the cancer is selected fromthe group consisting of: a non-hematologic malignancy including such asfor example, lung cancer, pancreatic cancer, breast cancer, ovariancancer, prostate cancer, head and neck cancer, gastric cancer,gastrointestinal cancer, colorectal cancer, esophageal cancer, cervicalcancer, liver cancer, kidney cancer, biliary duct cancer, gallbladdercancer, bladder cancer, sarcoma, brain cancer, central nervous systemcancer, and melanoma; and a hematologic malignancy such as for example,a leukemia, a lymphoma and other B cell malignancies, myeloma and otherplasma cell dysplasias or dyscrasias. In some embodiments, the cancer isselected from the group consisting of: breast cancer, head and neckcancer, lung cancer, stomach cancer, osteosarcoma, Non-Hodgkin'slymphoma (NEIL), acute lymphoblastic leukemia (ALL), mycosis fungoides(cutaneous T-cell lymphoma) choriocarcinoma, and chorioadenoma,nonleukemic meningeal cancer, soft tissue sarcoma (desmoid tumors,aggressive fibromatosis, bladder cancer, and central Nervous System(CNS) lymphoma. In some embodiments, the cancer is selected from thegroup consisting of: colorectal cancer, breast cancer, gastric cancer(e.g., stomach cancer), pancreatic cancer, liver cancer, lung cancer(e.g., non-small cell lung cancer and/or adenocarcinoma), head and neckcancer, ovarian cancer, gallbladder cancer, and basal cell cancer. Inparticular embodiments, the cancer is colorectal cancer.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomecomprising polyglutamated alpha tetrahydrofolate (e.g., an Lp-αPTHF suchas, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF) to asubject having or at risk of having cancer. In some embodiments, theliposome is pegylated. In some embodiments, the liposome is notpegylated. In additional embodiments, the liposome comprises a targetingmoiety that has a specific affinity for an epitope of an antigen on thesurface of a cancer cell. In additional embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeof a cell surface antigen selected from the group consisting of: GONMB,TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin,fibronectin extra-domain B (ED-B), VEGFR2 (CD309), tenascin, collagenIV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and Musk. This also includes the use of cancerstem cell targeting moieties such as those targeting CD 34, CD133 andCD44, CD138, and CD15. In some embodiments, the liposome comprises atargeting moiety that has specific affinity for an epitope of a cellsurface antigen(s) derived from or determined to be expressed on aspecific subject's tumor such as a neoantigen. In some embodiments, thetargeting moiety is an antibody or an antigen binding antibody fragment.In some embodiments, the liposome comprises αPTHF containing 4, 5, 2-10,4-6, or more than 5, glutamyl groups. In some embodiments, theadministered liposome comprises a αPTHF selected from: (a)polyglutamated 5-formyl-THF (e.g., polyglutamated [6S]-5-formyl-THF);(b) polyglutamated 10-formyl-THF (e.g., polyglutamated[6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF (e.g.,polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF(e.g., polyglutamated [6S]-5-methyl-THF); (e) polyglutamatedTetrahydrofolate THF (e.g., polyglutamated [6S]-Tetrahydrofolate THF);(f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated[6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g.,polyglutamated [6S]-5-formimino-THF). In some embodiments, theadministered liposome comprises polyglutamated 5,10-methylene-THF. Infurther embodiments, the administered liposome comprises polyglutamated[6R]-5,10-methylene-THF. In other embodiments, the administered liposomecomprises polyglutamated [6R,S]-5,10-methylene-THF. In some embodiments,the administered liposome comprises polyglutamated 5-methyl-THF. Infurther embodiments, the administered liposome comprises[6S]-5-methyl-THF. In other embodiments, the administered liposomecomprises [6R,S]-5-methyl-THF. In some embodiments, the administeredliposome comprises polyglutamated 5-formyl-THF. In further embodiments,the administered liposome comprises polyglutamated [6S]-5-formyl-THF. Inother embodiments, the administered liposome comprises polyglutamated[6R,S]-5-formyl-THF. In some embodiments the administered liposomalcomposition comprises tetraglutamated αPTHF. In some embodiments theadministered liposomal composition comprises pentaglutamated αPTHF. Insome embodiments the administered liposomal composition compriseshexaglutamated αPTHF. In some embodiments, a liposome of theadministered liposomal composition comprises αPTHF containing 4, 5,2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, aliposome of the administered liposomal composition comprises a αPTHFcontaining 1, 2, 3, or more than 3, glutamyl groups having a gammalinkage. In some embodiments, the cancer is selected from the groupconsisting of: lung (e.g., non-small lung cancer), pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, head and neck cancer,gastric cancer, gastrointestinal cancer, colorectal cancer, esophagealcancer, cervical cancer, liver cancer, kidney cancer, biliary ductcancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, melanoma,and a hematologic malignancy (e.g., a leukemia or lymphoma). In yetfurther embodiments, the cancer cell is a primary cell or a cell from acell line obtained/derived from a cancer selected from colorectalcancer, breast cancer, gastric cancer (e.g., stomach cancer), pancreaticcancer, liver cancer, lung cancer (e.g., non-small cell lung cancerand/or adenocarcinoma), head and neck cancer, ovarian cancer,gallbladder cancer, and basal cell cancer. In particular embodiments,the cancer cell is a primary cell or a cell from a cell lineobtained/derived from colorectal cancer.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering to a subject having or at risk ofhaving cancer, an effective amount of a liposomal composition comprisinga liposome that comprises polyglutamated alpha tetrahydrofolate and atargeting moiety that has a specific affinity for an epitope of anantigen on the surface of the cancer. In some embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeof a cell surface antigen selected from the group consisting of: GONMB,TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin,fibronectin extra-domain B (ED-B), VEGFR2 (CD309), tenascin, collagenIV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, the liposomecomprises a targeting moiety that a targeting moiety that has specificaffinity for an epitope of a cell surface antigen(s) derived from, ordetermined to be expressed on, a specific subject's cancer (tumor) suchas a neoantigen. In some embodiments, the targeting moiety is anantibody or an antigen binding antibody fragment. In some embodiments,the liposome comprises αPTHF containing 4, 5, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, the administered liposomecomprises αPTHF containing 4 glutamyl groups. In some embodiments, theadministered liposome comprises αPTHF containing 5 glutamyl groups. Insome embodiments, the administered liposome comprises αPTHF containing 6glutamyl groups. In some embodiments, the administered liposomecomprises a αPTHF selected from: (a) polyglutamated 5-formyl-THF (e.g.,polyglutamated [6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF(e.g., polyglutamated [6R]-10-formyl-THF); (c) polyglutamated5,10-methenyl-THF (e.g., polyglutamated [6R]-5,10-methenyl-THF); (d)polyglutamated 5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF);(e) polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, the administered liposome comprises polyglutamated5,10-methylene-THF. In further embodiments, the administered liposomecomprises polyglutamated [6R]-5,10-methylene-THF. In other embodiments,the administered liposome comprises polyglutamated[6R,S]-5,10-methylene-THF. In some embodiments, the administeredliposome comprises polyglutamated 5-methyl-THF. In further embodiments,the administered liposome comprises [6S]-5-methyl-THF. In otherembodiments, the administered liposome comprises [6R,S]-5-methyl-THF. Insome embodiments, the administered liposome comprises polyglutamated5-formyl-THF. In further embodiments, the administered liposomecomprises polyglutamated [6S]-5-formyl-THF. In other embodiments, theadministered liposome comprises polyglutamated [6R,S]-5-formyl-THF. Insome embodiments, the liposome comprises a αPTHF containing 1, 2, 3, ormore than 3, glutamyl groups having a gamma linkage.

In some embodiments, the administered liposomal composition comprisespegylated liposomes (e.g., TPLp-αPTHF). In some embodiments, theadministered liposomal composition comprises liposomes that are notpegylated. In some embodiments, liposomes of the administered liposomalcomposition comprise a αPTHF containing 4, 5, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, the administered liposomecomprises αPTHF containing 4 glutamyl groups. In some embodiments, theadministered liposome comprises αPTHF containing 5 glutamyl groups. Insome embodiments, the administered liposome comprises αPTHF containing 6glutamyl groups. In some embodiments, the administered liposomecomprises a αPTHF selected from: (a) polyglutamated 5-formyl-THF (e.g.,polyglutamated [6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF(e.g., polyglutamated [6R]-10-formyl-THF); (c) polyglutamated5,10-methenyl-THF (e.g., polyglutamated [6R]-5,10-methenyl-THF); (d)polyglutamated 5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF);(e) polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, the administered liposome comprises polyglutamated5,10-methylene-THF. In further embodiments, the administered liposomecomprises polyglutamated [6R]-5,10-methylene-THF. In other embodiments,the administered liposome comprises polyglutamated[6R,S]-5,10-methylene-THF. In some embodiments, the administeredliposome comprises polyglutamated 5-methyl-THF. In further embodiments,the administered liposome comprises [6S]-5-methyl-THF. In otherembodiments, the administered liposome comprises [6R,S]-5-methyl-THF. Insome embodiments, the administered liposome comprises polyglutamated5-formyl-THF. In further embodiments, the administered liposomecomprises polyglutamated [6S]-5-formyl-THF. In other embodiments, theadministered liposome comprises polyglutamated [6R,S]-5-formyl-THF. Insome embodiments, a liposome of the administered liposomal compositioncomprises a αPTHF containing 1, 2, 3, or more than 3, glutamyl groupshaving a gamma linkage. In some embodiments, the liposomal compositionis administered to treat a cancer selected from the group consisting of:lung cancer (e.g., non-small cell), pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, head and neck cancer, gastric cancer,gastrointestinal cancer, colorectal cancer, esophageal cancer, cervicalcancer, liver cancer, kidney cancer, biliary duct cancer, gallbladdercancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer,central nervous system cancer, melanoma, myeloma, a leukemia and alymphoma. In some embodiments, the liposomal composition is administeredto treat a cancer selected from the group consisting of: colorectalcancer, breast cancer, gastric cancer (e.g., stomach cancer), pancreaticcancer, liver cancer, lung cancer (e.g., non-small cell lung cancerand/or adenocarcinoma), head and neck cancer, ovarian cancer,gallbladder cancer, and basal cell cancer. In particular embodiments,the liposomal composition is administered to treat colorectal cancer.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomalcomposition to a subject having or at risk of having a cancer thatexpresses folate receptor on its cell surface, wherein the liposomalcomposition comprises liposomes that comprise (a) polyglutamated alphatetrahydrofolate (αPTHF) and (b) a targeting moiety that has specificbinding affinity for a folate receptor. In some embodiments, thetargeting moiety has specific binding affinity for folate receptor alpha(FR-α), folate receptor beta (FR-β), and/or folate receptor delta(FR-δ). In some embodiments, the targeting moiety has a specific bindingaffinity for folate receptor alpha (FR-α), folate receptor beta (FR-β),and/or folate receptor delta (FR-δ). In some embodiments, the targetingmoiety has a specific binding affinity for folate receptor alpha (FR-α)and folate receptor beta (FR-β). In some embodiments, the administeredliposomal composition comprises pegylated liposomes (e.g., TPLp-αPTHF).In some embodiments, the administered liposomal composition comprisesliposomes that are not pegylated. In some embodiments, liposomes of theadministered liposomal composition comprises a αPTHF containing 4, 5,2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, theadministered liposome comprises αPTHF containing 4 glutamyl groups. Insome embodiments, the administered liposome comprises αPTHF containing 5glutamyl groups. In some embodiments, the administered liposomecomprises αPTHF containing 6 glutamyl groups. In some embodiments, theadministered liposome comprises a αPTHF selected from: (a)polyglutamated 5-formyl-THF (e.g., polyglutamated [6S]-5-formyl-THF);(b) polyglutamated 10-formyl-THF (e.g., polyglutamated[6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF (e.g.,polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF(e.g., polyglutamated [6S]-5-methyl-THF); (e) polyglutamatedTetrahydrofolate THF (e.g., polyglutamated [6S]-Tetrahydrofolate THF);(f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated[6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g.,polyglutamated [6S]-5-formimino-THF). In some embodiments, theadministered liposome comprises polyglutamated 5,10-methylene-THF. Infurther embodiments, the administered liposome comprises polyglutamated[6R]-5,10-methylene-THF. In other embodiments, the administered liposomecomprises polyglutamated [6R,S]-5,10-methylene-THF. In some embodiments,the administered liposome comprises polyglutamated 5-methyl-THF. Infurther embodiments, the administered liposome comprises[6S]-5-methyl-THF. In other embodiments, the administered liposomecomprises [6R,S]-5-methyl-THF. In some embodiments, the administeredliposome comprises polyglutamated 5-formyl-THF. In further embodiments,the administered liposome comprises polyglutamated [6S]-5-formyl-THF. Inother embodiments, the administered liposome comprises polyglutamated[6R,S]-5-formyl-THF. In some embodiments, a liposome of the administeredliposomal composition comprises 1, 2, 3, or more than 3 glutamyl groupscontaining a gamma linkage. In some embodiments, the liposomalcomposition is administered to treat a cancer selected from the groupconsisting of: a non-hematologic malignancy including such as forexample, lung cancer, pancreatic cancer, breast cancer, ovarian cancer,prostate cancer, head and neck cancer, gastric cancer, gastrointestinalcancer, colorectal cancer, esophageal cancer, cervical cancer, livercancer, kidney cancer, biliary duct cancer, gallbladder cancer, bladdercancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervoussystem cancer, and melanoma; and a hematologic malignancy such as forexample, a leukemia, a lymphoma and other B cell malignancies, myelomaand other plasma cell dysplasias or dyscrasias. In some embodiments, theliposomal composition is administered to treat a cancer is selected fromthe group consisting of: breast cancer, head and neck cancer, lungcancer, stomach cancer, osteosarcoma, Non-Hodgkin's lymphoma (NHL),acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-celllymphoma) choriocarcinoma, and chorioadenoma, nonleukemic meningealcancer, soft tissue sarcoma (desmoid tumors, aggressive fibromatosis,bladder cancer, and central Nervous System (CNS) lymphoma. In someembodiments the liposomal composition is administered to treat a cancerselected from the group consisting of: colorectal cancer, breast cancer,gastric cancer (e.g., stomach cancer), pancreatic cancer, liver cancer,lung cancer (e.g., non-small cell lung cancer and/or adenocarcinoma),head and neck cancer, ovarian cancer, gallbladder cancer, and basal cellcancer.

In additional embodiments, the disclosure provides a method for cancermaintenance therapy that comprises administering an effective amount ofa liposomal composition comprising liposomes that contain polyglutamatedalpha tetrahydrofolate (Lp-αPTHF) to a subject that is undergoing or hasundergone cancer therapy. In some embodiments, the administeredliposomal composition is a PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHFor TPLp-αPTHF. In some embodiments, the administered liposomalcomposition comprises pegylated liposomes (e.g., PLp-αPTHF, NTPLp-αPTHF,or TPLp-αPTHF). In some embodiments, the administered liposomalcomposition comprises targeted liposomes (e.g., TLp-αPTHF orTPLp-αPTHF). In some embodiments, the administered liposomal compositioncomprises liposomes that are pegylated and comprise a targeting moiety(e.g., TPLp-αPTHF). In some embodiments, a liposome of the administeredliposomal composition comprises polyglutamated alpha tetrahydrofolatethat contains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the administered liposome comprises αPTHF containing 4glutamyl groups. In some embodiments, the administered liposomecomprises αPTHF containing 5 glutamyl groups. In some embodiments, theadministered liposome comprises αPTHF containing 6 glutamyl groups. Insome embodiments, the administered liposome comprises a αPTHF selectedfrom: (a) polyglutamated 5-formyl-THF (e.g., polyglutamated[6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF (e.g.,polyglutamated [6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF(e.g., polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF); (e)polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, the administered liposome comprises polyglutamated5,10-methylene-THF. In further embodiments, the administered liposomecomprises polyglutamated [6R]-5,10-methylene-THF. In other embodiments,the administered liposome comprises polyglutamated[6R,S]-5,10-methylene-THF. In some embodiments, the administeredliposome comprises polyglutamated 5-methyl-THF. In further embodiments,the administered liposome comprises [6S]-5-methyl-THF. In otherembodiments, the administered liposome comprises [6R,S]-5-methyl-THF. Insome embodiments, the administered liposome comprises polyglutamated5-formyl-THF. In further embodiments, the administered liposomecomprises polyglutamated [6S]-5-formyl-THF. In other embodiments, theadministered liposome comprises polyglutamated [6R,S]-5-formyl-THF. Insome embodiments, a liposome of the administered liposomal compositioncomprises 1, 2, 3, or more than 3 glutamyl groups containing a gammalinkage.

In additional embodiments, the disclosure provides a method for treatinga disorder of the immune system that comprises administering aneffective amount of a liposomal composition comprising liposomes thatcontain polyglutamated alpha tetrahydrofolate (e.g., Lp-αPTHF,PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF or TPLp-αPTHF) to asubject having or at risk of having a disorder of the immune system. Insome embodiments, the liposomal composition is administered to treat anautoimmune disease. In a further embodiment, the liposomal compositionis administered to treat rheumatoid arthritis. In another embodiment,the liposomal composition is administered to treat inflammation. In someembodiments, the administered liposomal composition comprises pegylatedliposomes (e.g., PLp-αPTHF, NTPLp-αPTHF, or TPLp-αPTHF). In someembodiments, the administered liposomal composition comprises targetedliposomes (e.g., TLp-αPTHF or TPLp-αPTHF) that contain a targetingmoiety having a specific affinity for a surface antigen on a target cellof interest (e.g., an immune cell). In further embodiments, theadministered liposomal composition comprises liposomes that arepegylated and comprise a targeting moiety (e.g., TPLp-αPTHF)). In someembodiments, a liposome of the administered liposomal compositioncomprises pentaglutamated alpha tetrahydrofolate that contains 4, 5,2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, theadministered liposome comprises αPTHF containing 4 glutamyl groups. Insome embodiments, the administered liposome comprises αPTHF containing 5glutamyl groups. In some embodiments, the administered liposomecomprises αPTHF containing 6 glutamyl groups. In some embodiments, theadministered liposome comprises a αPTHF selected from: (a)polyglutamated 5-formyl-THF (e.g., polyglutamated [6S]-5-formyl-THF);(b) polyglutamated 10-formyl-THF (e.g., polyglutamated[6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF (e.g.,polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF(e.g., polyglutamated [6S]-5-methyl-THF); (e) polyglutamatedTetrahydrofolate THF (e.g., polyglutamated [6S]-Tetrahydrofolate THF);(f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated[6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g.,polyglutamated [6S]-5-formimino-THF). In some embodiments, theadministered liposome comprises polyglutamated 5,10-methylene-THF. Infurther embodiments, the administered liposome comprises polyglutamated[6R]-5,10-methylene-THF. In other embodiments, the administered liposomecomprises polyglutamated [6R,S]-5,10-methylene-THF. In some embodiments,the administered liposome comprises polyglutamated 5-methyl-THF. Infurther embodiments, the administered liposome comprises[6S]-5-methyl-THF. In other embodiments, the administered liposomecomprises [6R,S]-5-methyl-THF. In some embodiments, the administeredliposome comprises polyglutamated 5-formyl-THF. In further embodiments,the administered liposome comprises polyglutamated [6S]-5-formyl-THF. Inother embodiments, the administered liposome comprises polyglutamated[6R,S]-5-formyl-THF. In some embodiments, a liposome of the administeredliposomal composition comprises 1, 2, 3, or more than 3 glutamyl groupscontaining a gamma linkage.

The disclosure also provides a method of delivering polyglutamated alphatetrahydrofolate to a tumor and/or cancer cell that comprises:administering to a subject having the tumor, a composition comprisingpolyglutamated alpha tetrahydrofolate (L-αPTHF) and a targeting moietythat has a specific binding affinity for an epitope of a surface antigenon the tumor cell or cancer cell. In some embodiments, the administeredtargeting moiety is associated with a delivery vehicle. In someembodiments, the delivery vehicle is an antibody or an antigen bindingfragment of an antibody. In further embodiments, the delivery vehicle isa liposome. In some embodiments, the antibody, antigen-binding antibodyfragment, or liposome is pegylated. In some embodiments, theadministered composition comprises polyglutamated alpha tetrahydrofolatethat contains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the administered composition comprises tetraglutamatedalpha tetrahydrofolate. In some embodiments, the administeredcomposition comprises pentaglutamated alpha tetrahydrofolate. In otherembodiments, the administered composition comprises hexaglutamated alphatetrahydrofolate. In some embodiments, the administered compositioncomprises a αPTHF selected from: (a) polyglutamated 5-formyl-THF (e.g.,polyglutamated [6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF(e.g., polyglutamated [6R]-10-formyl-THF); (c) polyglutamated5,10-methenyl-THF (e.g., polyglutamated [6R]-5,10-methenyl-THF); (d)polyglutamated 5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF);(e) polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, the administered composition comprises polyglutamated5,10-methylene-THF. In further embodiments, the administered compositioncomprises polyglutamated [6R]-5,10-methylene-THF. In other embodiments,the administered composition comprises polyglutamated[6R,S]-5,10-methylene-THF. In some embodiments, the administeredcomposition comprises polyglutamated 5-methyl-THF. In furtherembodiments, the administered composition comprises [6S]-5-methyl-THF.In other embodiments, the administered composition comprises[6R,S]-5-methyl-THF. In some embodiments, the administered compositioncomprises polyglutamated 5-formyl-THF. In further embodiments, theadministered composition comprises polyglutamated [6S]-5-formyl-THF. Inother embodiments, the administered composition comprises polyglutamated[6R,S]-5-formyl-THF.

In additional embodiments, the disclosure provides a method of preparinga liposomal composition that comprises a liposomal polyglutamated alphatetrahydrofolate (αPTHF) composition, the method comprising: forming amixture comprising: liposomal components and α polyglutamatedtetrahydrofolate in solution; homogenizing the mixture to form liposomesin the solution; and processing the mixture to form liposomes containingpolyglutamated tetrahydrofolate. In some embodiments, the polyglutamatedalpha tetrahydrofolate contains 4, 5, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, the αPTHF composition comprisespentaglutamated alpha tetrahydrofolate. In some embodiments, the αPTHFcomposition comprises tetraglutamated alpha tetrahydrofolate. In otherembodiments, the αPTHF composition comprises hexaglutamated alphatetrahydrofolate. In some embodiments, the polyglutamated alphatetrahydrofolate contains 1, 2, 3, or more than 3, glutamyl groupscontaining a gamma linkage. In some embodiments, the αPTHF compositioncontains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, glutamyl groupsin the D-form. In some embodiments, the αPTHF composition contains 2, 3,4, 5, 6, 7, 8, 9, 10, or more than 10, glutamyl groups in the L-form. Insome embodiments, the αPTHF composition contains 2, 3, 4, 5, or morethan 5, glutamyl groups in the L-form, and 1, 2, 3, 4, 5 or more than 5,glutamyl groups in the D-form. In some embodiments, compositioncomprises a αPTHF selected from: (a) polyglutamated 5-formyl-THF (e.g.,polyglutamated [6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF(e.g., polyglutamated [6R]-10-formyl-THF); (c) polyglutamated5,10-methenyl-THF (e.g., polyglutamated [6R]-5,10-methenyl-THF); (d)polyglutamated 5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF);(e) polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, the composition comprises polyglutamated5,10-methylene-THF. In further embodiments, the composition comprisespolyglutamated [6R]-5,10-methylene-THF. In other embodiments, thecomposition comprises polyglutamated [6R,S]-5,10-methylene-THF. In someembodiments, the composition comprises polyglutamated 5-methyl-THF. Infurther embodiments, the composition comprises [6S]-5-methyl-THF. Inother embodiments, the composition comprises [6R,S]-5-methyl-THF. Insome embodiments, the composition comprises polyglutamated 5-formyl-THF.In further embodiments, the composition comprises polyglutamated[6S]-5-formyl-THF. In other embodiments, the administered compositioncomprises polyglutamated [6R,S]-5-formyl-THF.

In one embodiment, the disclosure provides a kit comprising apolyglutamated alpha tetrahydrofolate composition and/or a αPTHFdelivery vehicle such as a liposome containing αPTHF or an αPTHFimmunoconjugate (e.g., an ADC) described herein.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIGS. 1A-1N show exemplary chemical formulas of alpha tetrahydrofolates:alpha 5-10 methylene THF (FIG. 1A), alpha 5-10 methylene THF diglutamate(FIG. 1B), alpha 5-10 methylene THF triglutamate (FIGS. 1C and 1D),alpha 5-10 methylene THF tetraglutamates (FIGS. 1E and 1F), alpha 5-10methylene THF pentaglutamates (FIGS. 1G and 111), alpha 5-10 methyleneTHF hexaglutamates (FIGS. 1I and 1J), alpha 5-10 methylene THFheptaglutamates (FIGS. 1K and 1L), alpha 5-10 methylene THFoctaglutamates (FIGS. 1M and 1N), and exemplary alpha tetrahydrofolatepolyglutamate derivatives of Tetrahydrofolate THF, 10 formyl THF, 5formyl THF, 5-methyl THF, 5 formimino THF, 5,10 methenyl THF, and 5,10methylene THF (FIGS. 1O-1Q). FIGS. 1R-1U present depictions of exemplarybranched 5-10 methylene THF polyglutamate structures, including abranched polyglutamate having an alpha glutamyl backbone and gammaglutamyl branches (FIG. 1S), a branched polyglutamate having a gammaglutamyl backbone and alpha glutamyl branches (FIG. 1T), and a branchedpolyglutamate having an alpha glutamyl backbone and both gamma glutamylbranches and alpha glutamyl branches (FIG. 1U).

FIG. 2 presents the relative potency of liposomal pemetrexed alpha-Lhexaglutamate (liposomal aG6) and its mirror image, liposomal alpha-Dhexaglutamate (liposomal aDG6) relative to pemetrexed following exposureof the cancer cell lines SW620 (CRC), HT-29 (colon cancer), H1806(triple negative breast cancer), OAW28 (ovarian cancer), H292 (NSCLC,adenocarcinoma subtype), and H2342 (NSCLC, adenocarcinoma subtype), over48 hours.

FIG. 3 presents an example dose response relationship of free pemetrexedL-gamma hexaglutamate (gG6), liposomal pemetrexed L-gamma hexaglutamate(liposomal gG6), pemetrexed, and folate receptor alpha targetingantibody (FR1Ab) liposomal pemetrexed L-gamma hexaglutamate (liposomalgG6-FR1Ab) in the NCI H2342 non-small cell lung cancer (NSCLC),adenocarcinoma subtype depicted as the percentage of viable cells after48 hours of treatment. Folate receptor alpha targeted liposomescontaining alpha polyglutamated pemetrexed are expected to also besuccessful in targeting and reducing the viability of NCI H2342non-small cell lung cancer cells.

FIG. 4 presents an example dose response relationship of free pemetrexedL-gamma hexaglutamate (gG6), liposomal pemetrexed L-gamma hexaglutamate(liposomal gG6), pemetrexed, and folate receptor alpha targetingantibody (FR1Ab) liposomal pemetrexed L-gamma hexaglutamate (liposomalgG6-FR1Ab) in the HT-29 (colon cancer) at 48 hours. Folate receptoralpha targeted liposomes containing alpha polyglutamated pemetrexed areexpected to also be successful in targeting and reducing the viabilityof HT-29 (colon cancer) cells.

FIG. 5 presents the treatment effect on HCC1806 triple negative breastcancer cells following exposure of liposomal pemetrexed alpha-Lhexaglutamate (Lps Hexa aG6), liposomal pemetrexed alpha-D hexaglutamate(Lps Hexa aDG6), and to pemetrexed over 48 hours.

FIG. 6 presents the treatment effect on OAW28 ovarian cancer cellsfollowing exposure of liposomal pemetrexed alpha-L hexaglutamate (LpsHexa aG6), liposomal pemetrexed alpha-D hexaglutamate (Lps Hexa aDG6),and to pemetrexed over 48 hours.

FIG. 7 presents the treatment effect on H292 non-small cell lung cancercells following exposure of liposomal pemetrexed alpha-L hexaglutamate(Lps Hexa aG6), liposomal pemetrexed alpha-D hexaglutamate (Lps HexaaDG6), as compared to pemetrexed over 48 hours.

FIG. 8 presents the treatment effect on H292 non-small cell lung cancercells following exposure of various dose levels ranging from 16 to 128nM of liposomal pemetrexed alpha-L hexaglutamate (Liposomal aG6),liposomal pemetrexed alpha-D hexaglutamate (Liposomal aDG6), andpemetrexed over 48 hours. At each of the tested dose ranges, theliposomal pemetrexed aG6 formulation is superior to inhibiting H292non-small cell lung cancer cells compared to pemetrexed.

FIG. 9 presents the treatment effect on HCC1806 triple negative breastcancer cells following exposure of various dose levels ranging from 16to 128 nM of liposomal pemetrexed alpha-L hexaglutamate (Liposomal aG6),liposomal pemetrexed alpha-D hexaglutamate (Liposomal aDG6), andpemetrexed over 48 hours. At each of the tested doses, the liposomalpemetrexed aG6 formulation is superior to pemetrexed in inhibitingHCC1806 triple negative breast cancer cells.

FIG. 10 presents the treatment effect on OAW28 ovarian cancer cells ofliposomal pemetrexed alpha-L hexaglutamate (Liposomal aG6), liposomalalpha-D hexaglutamate (Liposomal aDG6), and pemetrexed followingexposure over 48 hours following exposure over a range ofconcentrations. At the dose of 128 nM, pemetrexed appears to moreeffective than the Liposomal pemetrexed aG6 liposomal formulation,whereas the liposomal formulation at the dose of 32 nM and 64 nM has abetter treatment effect than pemetrexed; at 16 nM the Liposomalpemetrexed aG6 treatment effect is similar in to pemetrexed.

FIG. 11 shows the toxicity of liposomal pemetrexed alpha-L hexaglutamate(Liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (LiposomalaDG6), and pemetrexed on differentiating human neutrophils at 64 nM, 128nM, and 264 nM. The figure demonstrates that liposomal pemetrexed aG6 issignificantly less toxic to differentiating human neutrophils thanpemetrexed.

FIG. 12 shows the effect of liposomal pemetrexed alpha-L hexaglutamate(liposomal aG6), liposomal alpha-D hexaglutamate (liposomal aDG6), andpemetrexed on neutrophils (differentiated from CD34+ cells) followingexposure of various dose levels ranging from 16 to 128 nM of thecorresponding agent over 48 hours.

FIG. 13 shows the effect of liposomal pemetrexed alpha-L hexaglutamate(liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomalaDG6), and pemetrexed on AML12 liver cells following exposure over 48hours at 16 nM, 32 nM, and 64 nM, and 128 nM of the corresponding agent.Strikingly, there does not appear to be any toxicity to the AML livercells following treatment with a liposomal pemetrexed aG6 at any of theliposomal agents at the dose levels tested. In contrast, pemetrexedtreatment results in a reduction in the AML12 liver cell counts ofapproximately 40% at all doses studied.

FIG. 14 shows the effect of liposomal pemetrexed alpha-L hexaglutamate(liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomalaDG6), and pemetrexed on CCD841 colon epithelium cells followingexposure over 48 hours at 16 nM, 32 nM, and 64 nM, and 128 nM, of thecorresponding agent. At all of the concentrations tested, pemetrexedleads to approximately a ≥50% decrease in the number of CCD841 colonepithelium cells compared to approximately a 20% or less decrease incell number after treatment with each of the liposome compositionstested.

FIG. 15 depicts the structure of polyglutamate antifolate, Cisplatin(CDDP) and two potential aG6-Cisplatin complexes. The pH dependentformation of the interstrand and/or instrastrand coordination betweenthe carboxyl groups of the polyglutamated antifolate and cisplatin islikely to disassemble into individual molecules of aG6 and cisplatinupon encountering acidic pH of lysosomes (pH 4-5) and presence ofchloride ions inside the cells.

FIG. 16 presents the effects of liposomal aG6 treatment of mice with 40mg/kg and 80 mg/kg given once weekly for 4 weeks upon the hematologicparameters: white blood cell (WBC) counts, neutrophil counts and asplatelet counts. No appreciable decrease in mean neutrophil, mean whiteblood cell or mean platelet counts was observed.

FIG. 17 presents the effects of liposomal aG6 treatment of mice with 40mg/kg and 80 mg/kg given once weekly for 4 weeks upon hemoglobin andreticulocyte indices. There is a minimal decrease in mean hemoglobinconcentrations at the higher dose level. In parallel there is a slightincrease in mean reticulocytosis indices

FIG. 18 presents the effects of liposomal aG6 treatment of mice with 40mg/kg and 80 mg/kg given once weekly for 4 weeks upon hepatic markersincluding serum aspartate transaminase (AST) and serum alaninetransaminase (ALT) along with serum albumin. There was no appreciableincreases in liver transaminases mean AST or mean ALT levels and therewas no observed change in mean albumin levels.

FIG. 19 presents the relative tumor volume of immunodeficient femaleNu/J mice (6-8 weeks old) inoculated with NCI-H292 (Non-Small Cell LungCancer) cells and administered control, pemetrexed, and Liposomal aG6intravenously at 167 mg/kg once every three weeks. As can be seen fromthese preliminary data, liposomal aG6 provides reduced tumor controlcompared to pemetrexed.

FIGS. 20A-F present the dose response relationship of liposomalpemetrexed alpha-L triglutamate (Liposomal aG3), liposomal pemetrexedalpha-L pentaglutamate (Liposomal aG5), liposomal pemetrexed alpha-Loctaglutamate (Liposomal aG7), and a combination of liposomal pemetrexedalpha-L hexaglutamate (aG6) and alpha-L dodecaglutamate (aG12)(Liposomal aG6 and aG12), over 48 hours on H2342 (NSCLC, adenocarcinomasubtype) (FIG. 20A), H292 (NSCLC, adenocarcinoma subtype) (FIG. 20B),HT-29 (colon cancer) (FIG. 20C), HCC1806 (triple negative breast cancer)(FIG. 20D), MCF7 (ER+ breast cancer) (FIG. 20E), and OAW28 (ovariancancer) (FIG. 20F). Cell viability was determined by CellTiter-Glo®(CTG) luminescent cell viability assay essentially as described inExample 1. As shown in all cell lines, the potency of each of thepolyglutamated pemetrexed liposomal compositions well exceeded that ofthe liposomal vehicle and empty liposome controls.

DETAILED DESCRIPTION

The disclosure generally relates to polyglutamated alphatetrahydrofolate compositions. The compositions provide advances overprior treatments of hyperproliferative diseases such as cancer. Methodsof making, delivering and using the polyglutamated alphatetrahydrofolate compositions are also provided. The polyglutamatedalpha compositions have uses that include but are not limited totreating (e.g., treating and/or preventing) hyperproliferative diseasessuch as cancer, disorders of the immune system such as inflammation andrheumatoid arthritis, and infectious disease such as HIV and malaria.The polyglutamated alpha compositions also have uses in combinationtherapy with one or more therapeutic agents such as a chemotherapy drug(e.g., 5-fluorouracil) to enhance the effectiveness of the therapeuticagent or as a “chemoprotectant” (e.g., in combination with an antifolatesuch as methotrexate) to reduce toxic side effects associated with thetherapeutic agent(s).

I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure pertains.

It is understood that wherever embodiments, are described herein withthe language “comprising” otherwise analogous embodiments, described interms of “containing” “consisting of” and/or “consisting essentially of”are also provided. However, when used in the claims as transitionalphrases, each should be interpreted separately and in the appropriatelegal and factual context (e.g., in claims, the transitional phrase“comprising” is considered more of an open-ended phrase while thetransitional phrases “consisting of” is more exclusive and “consistingessentially of” achieves a middle ground).

As used herein, the singular form “a”, “an”, and “the”, includes pluralreferences unless it is expressly stated or is unambiguously clear fromthe context that such is not intended.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both A and B; A or B; A (alone); and B (alone).Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C”is intended to encompass each of the following embodiments: A, B, and C;A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A(alone); B (alone); and C (alone).

Headings and subheadings are used for convenience and/or formalcompliance only, do not limit the subject technology, and are notreferred to in connection with the interpretation of the description ofthe subject technology. Features described under one heading or onesubheading of the subject disclosure may be combined, in variousembodiments, with features described under other headings orsubheadings. Further it is not necessarily the case that all featuresunder a single heading or a single subheading are used together inembodiments.

The terms “tetrahydrofolate” and “THF” are used interchangeably toinclude a salt, acid and and/or free base form of a tetrahydrofolate(e.g., tetrahydrofolate disodium). Unless otherwise expressly stated orunambiguously clear from the context, “THF(s)” and “tetrahydrofolate(s)”include natural and unnatural THF forms, including onecarbon-substituted THF derivatives. In particular, unless otherwiseexpressly stated or unambiguously clear from the context, “THF(s)” and“tetrahydrofolate(s)” include diasteromeric compositions having a [6R]configuration at the C-6 atom of the tetrahydropterin component of theTHF, diasteromeric compositions having a [6S] configuration at the C-6atom, and/or mixtures of [6,R,S] diastereomers (e.g., 1:1). Unlessotherwise expressly stated or unambiguously clear from the context,“THF(s)” and “tetrahydrofolate(s)” include: (a) 5-formyl-THF (e.g.,[6S], [6R,S], or [6R]-5-formyl-THF); (b) 5-formyl-THF (e.g., [6S],[6R,S], or [6R]-5-formyl-THF); (c) 5,10-methenyl-THF (e.g., [6R],[6R,S], or [6S], -5,10-methenyl-THF); (d) 5-methyl-THF (e.g., [6S],[6R,S], or [6R], -5-methyl-THF); (e) Tetrahydrofolate THF((2S)-2-{[4-({[2-amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl]methyl}amino)phenyl]formamido} pentanedioic acid) ((e.g., [6S], [6R,S], and[6R,]-((2S)-2-{[4-({[2-amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl]methyl}amino)phenyl]formamido}pentanedioic acid)); (f) 5,10-methylene-THF (e.g., [6R],[6R,S], or [SR], -5,10-methylene-THF); and (g) 5-formimino-THF (e.g.,[6S], [6R,S], or [6R], -5-formimino-THF. In some embodiments, thedisclosure provides a composition containing a THF diastereomer selectedfrom (a) [6S]-5-formyl-THF; (b) [6R]-5-formyl-THF; (c)[6R]-5,10-methenyl-THF; (d) [6S]-5-methyl-THF; (e) [6S]-TetrahydrofolateTHF; (f) [6R]-5,10-methylene-THF; and (g) [6S]-5-formimino-THF. In someembodiments, the disclosure provides a composition containing a THFdiastereomer mixture (e.g., a diastereoisomeric mixture[6R,S]-5-methyl-THF (1:1), and/or a diastereoisomeric mixture[6R,S]-5-CHO-THF (1:1) Compositions containing a THF salt may furthercontain any of a variety of cations, such as Na⁺, Mg²⁺, K⁺, NH₄ ⁺,and/or Ca²⁺. In particular embodiments, the salts are pharmaceuticallyacceptable salts. In additional particular embodiments, the THF saltcontains Nat Tetrahydrofolate contains one L-gamma glutamyl group, andis therefore considered to be monoglutamated for the purpose of thisdisclosure.

The term “Tetrahydrofolate THF” specifically refers to a THF compositionhaving the structure2-{[4-({[(6S)-2-amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl]methyl}amino) phenyl] formamido} pentanedioic acid. A “Tetrahydrofolate THF”may also be referred to herein as a species of tetrahydrofolate (THF).

The terms “polyglutamate”, polyglutamated”, or variations thereof, referto a composition comprising at least one chain of 2 or more linkedglutamyl groups. Polyglutamate chains can be linear or branched. Linearpolyglutamate chains can contain for example, glutamyl groups containingeither an alpha carboxyl group or a gamma carboxyl group linkage.Branched polyglutamate chains can comprise for example, one or moreglutamyl groups that contain both an alpha carboxyl group and a gammacarboxyl group linkage to other glutamyl groups, thereby providing abranch point of the polyglutamate. Exemplary branched polyglutamates aredepicted in FIGS. 1R-1U. Polyglutamate chains comprise an N-terminalglutamyl group and one or more C-terminal glutamyl groups. TheN-terminal glutamyl group of a polyglutamate chain is not linked toanother glutamyl group via its amine group, but is linked to one or moreglutamyl group via its carboxylic acid group. In some embodiments, theN-terminal glutamyl group of a polyglutamated-tetrahydrofolate is theglutamyl group of tetrahydrofolate. The C-terminal glutamyl group orgroups of a polyglutamate chain are linked to another glutamyl group viatheir amine group, but are not linked to another glutamyl group viatheir carboxylic acid group.

The terms “polyglutamated-tetrahydrofolate”, “polyglutamated-THF”,“THF-PG”, “PTHF” and iterations thereof, are used interchangeably hereinto refer to a tetrahydrofolate composition that comprises at least oneglutamyl group in addition to the glutamyl group of tetrahydrofolate(i.e., THF-PG_(n), wherein n≥1). Reference to the number of glutamylgroups in a αPTHF (αTHF-PG) herein takes into account the glutamyl groupof tetrahydrofolate. For example, a THF-PG composition containing 5glutamyl residues in addition to the glutamyl group of THF is referredto herein as hexaglutamated tetrahydrofolate or tetrahydrofolatehexaglutamate. In some embodiments, polyglutamated-tetrahydrofolate, isa member selected from: (a) polyglutamated 5-formyl-THF; (b)polyglutamated 10-formyl-THF; (c) polyglutamated 5,10-methenyl-THF; (d)polyglutamated 5-methyl-THF; (e) polyglutamated tetrahydrofolate((2S)-2-{[4-({[2-amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl]methyl}amino)phenyl]formamido} pentanedioic acid) ((e.g., [6S], [6R,S], and[6R,]-((2S)-2-{[4-({[2-amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl]methyl}amino)phenyl] formamido} pentanedioic acid)); (f) polyglutamated5,10-methylene-THF; and (g) polyglutamated 5-formimino-THF. In furtherembodiments, the polyglutamated-tetrahydrofolate is a member selectedfrom: (a) polyglutamated [6S]-5-formyl-THF; (b) polyglutamated[6R]-10-formyl-THF; (c) polyglutamated[6R]-5,10-methenyl-THF; (d)polyglutamated [6S]-5-methyl-THF; (e) polyglutamated[6S]-Tetrahydrofolate THF; (f) polyglutamated [6R]-5,10-methylene-THF;and (g) polyglutamate[6S]-5-formimino-THF. In some embodiments, thepolyglutamated-tetrahydrofolate is [6R]-5,10-methylene-THF. In someembodiments, the polyglutamated-tetrahydrofolate is [6S]-5-methyl-THF.In some embodiments, the polyglutamated-tetrahydrofolate is[6S]-5-formyl-THF. In other embodiments, thepolyglutamated-tetrahydrofolate is a [6R,S]-5,10-methylene-THFdiastereomeric mixture, a [6R,S]-5-methyl-THF diastereomeric mixture, ora [6R,S]-5-formyl-THF diastereomeric mixture (e.g., 1:1 w/w).

The terms “alpha glutamyl group”, “alpha glutamate”, and “alpha linkage”as they relate to the linkage of a glutamyl group, refers to a glutamylgroup that contains an alpha carboxyl group linkage. In someembodiments, the alpha linkage is an amide bond between the alphacarboxyl group of one glutamyl group and a second glutamyl group. Thealpha linkage can be between a glutamyl group and the glutamyl group oftetrahydrofolate, or between the glutamyl group and a second glutamylgroup that is not present in tetrahydrofolate, such as a glutamyl groupwithin a polyglutamate chain attached to tetrahydrofolate. In someembodiments, an “alpha glutamyl group” of a provided polyglutamatedalpha tetrahydrofolate has both an alpha carboxyl group linkage and agamma carboxyl group linkage. In some embodiments, the alpha glutamylgroup is in the L-form. In some embodiments, the alpha glutamyl group isin the D-form. In some embodiments, the glutamyl group is in the L-form.In some embodiments, one or more glutamyl groups in the polyglutamatedalpha tetrahydrofolate are in the L form and one or more glutamyl groupsin the polyglutamated alpha tetrahydrofolate are in the D form.

The terms “polyglutamated alpha tetrahydrofolate”, “α-polyglutamatedtetrahydrofolate”, “αPTHF”, “polyglutamated alpha-tetrahydrofolate”,“polyglutamated alpha THF”, “αTHF-PG”, and iterations thereof, are usedinterchangeably herein to refer to a tetrahydrofolate composition thatcomprises at least one glutamyl group having an alpha carboxyl grouplinkage (e.g., THF-PG_(n), wherein n≥1 α glutamyl group). Reference tothe number of glutamyl groups in a αPTHF (αTHF-PG) herein takes intoaccount the glutamyl group of tetrahydrofolate. For example, a αTHF-PGcomposition containing 5 glutamyl groups in addition to the glutamylgroup of THF, and wherein at least one of the glutamyl groups has aalpha carboxyl linkage, may be referred to herein as alphahexaglutamated tetrahydrofolate, hexaglutamated alpha tetrahydrofolateor alpha tetrahydrofolate hexaglutamate.

The terms “gamma glutamyl group”, “gamma glutamate”, and “gammalinkage”, as they relate to the linkage of a glutamyl group, refers to aglutamyl group that contains a gamma carboxyl group linkage. In someembodiments, the gamma linkage is an amide bond between the gammacarboxyl group of one glutamyl group and a second glutamyl group. Thegamma linkage can be between a glutamyl group and the glutamyl group oftetrahydrofolate, or between the glutamyl group and a second glutamylgroup that is not present in tetrahydrofolate, such as a glutamyl groupwithin a polyglutamate chain attached to tetrahydrofolate. In someembodiments, one or more gamma linked glutamyl groups in thepolyglutamated alpha tetrahydrofolate is in the L form. In someembodiments, one or more gamma linked glutamyl groups in thepolyglutamated alpha tetrahydrofolate is in the D form. In someembodiments, one or more gamma linked glutamyl groups in thepolyglutamated alpha tetrahydrofolate is in the L form and one or moregamma linked glutamyl groups in the polyglutamated alphatetrahydrofolate is in the D form.

As use herein, the term “isolated” refers to a composition which is in aform not found in nature. Isolated polyglutamated alpha compositionsinclude those which have been purified to a degree that they are nolonger in a form in which they are found in nature. In some embodiments,a polyglutamated alpha tetrahydrofolate which is isolated issubstantially pure. Isolated compositions will be free or substantiallyfree of material with which they are naturally associated such as othercellular components such as proteins and nucleic acids with which theymay potentially be found in nature, or the environment in which they areprepared (e.g., cell culture). The polyglutamated alpha compositions maybe formulated with diluents or adjuvants and still for practicalpurposes be isolated—for example, the polyglutamated alpha compositionswill normally be mixed with pharmaceutically acceptable carriers ordiluents when used in diagnosis or therapy. In some embodiments, theisolated polyglutamated alpha compositions (e.g., alpha polyglutamatesand delivery vehicles such as liposomes containing the alphapolyglutamate contain less than 1% or less than 0.1% undesired DNA orprotein content. In some embodiments, the alpha polyglutamatecompositions (e.g., alpha polyglutamate and delivery vehicles such asliposomes containing the alpha polyglutamate) are “isolated.”

The term “targeting moiety” is used herein to refer to a molecule thatprovides an enhanced affinity for a selected target, e.g., a cell, celltype, tissue, organ, region of the body, or a compartment, e.g., acellular, tissue or organ compartment. The targeting moiety can comprisea wide variety of entities. Targeting moieties can include naturallyoccurring molecules, or recombinant or synthetic molecules. In someembodiments, the targeting moiety is an antibody, antigen-bindingantibody fragment, bispecific antibody or other antibody-based moleculeor compound. In some embodiments, the targeting moiety is an aptamer,avimer, a receptor-binding ligand, a nucleic acid, a biotin-avidinbinding pair, a peptide, protein, carbohydrate, lipid, vitamin, toxin, acomponent of a microorganism, a hormone, a receptor ligand or anyderivative thereof. Other targeting moieties are known in the art andare encompassed by the disclosure.

The terms “specific affinity”, “specifically binds”, and “enhancedaffinity”, mean that a targeting moiety such as an antibody or antigenbinding antibody fragment, reacts or associates more frequently, morerapidly, with greater duration, with greater affinity, or with somecombination of the above to the epitope, protein, or target moleculethan with alternative substances, including proteins unrelated toantigens containing the target epitope. Because of the sequence identitybetween homologous proteins in different species, specific affinity can,in several embodiments, include a binding agent that recognizes anepitope on a protein and/or target molecule in more than one species.Likewise, because of homology within certain regions of polypeptidesequences of different proteins, the term “specific affinity” or“specifically binds” can include a binding agent that recognizes anepitope that is present on more than one protein and/or target molecule.It is understood that, in certain embodiments, a targeting moiety thatspecifically binds a first target may or may not specifically bind asecond target. As such, “specific affinity” does not necessarily require(although it can include) exclusive binding, e.g., binding to an epitopeon a single target. Thus, a targeting moiety may, in certainembodiments, specifically bind an epitope that is present on more thanone target. In certain embodiments, multiple targets may be bound by thesame targeting moiety specifically binds an epitope that is present onmultiple targets.

The term “epitope” refers to that portion of an antigen capable of beingrecognized and specifically bound by a targeting moiety (i.e., bindingmoiety) such as an antibody. When the antigen is a polypeptide, epitopescan be formed both from contiguous amino acids and noncontiguous aminoacids juxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained upon protein denaturing,whereas epitopes formed by tertiary folding are typically lost uponprotein denaturing. An epitope typically includes at least 3, and moreusually, at least 5 or 8-10 amino acids in a unique spatialconformation.

Expressions like “binding affinity for a target”, “binding to a target”,“enhanced affinity”, and analogous expressions known in the art refer toa property of a targeting moiety which may be directly measured throughthe determination of the affinity constants, e.g., the amount oftargeting moiety that associates and dissociates at a given antigenconcentration. Different methods can be used to characterize themolecular interaction, such as, but not limited to, competitionanalysis, equilibrium analysis and microcalorimetric analysis, andreal-time interaction analysis based on surface plasmon resonanceinteraction (for example using a Biacore® instrument). These methods arewell-known to the skilled person and are described, for example, in Neriet al., Tibtech 14:465-470 (1996), and Jansson et al., J. Biol. Chem.272:8189-8197 (1997).

The term “delivery vehicle” refers generally to any compositions thatacts to assist, promote or facilitate entry of polyglutamated alphatetrahydrofolate into a cell. Such delivery vehicles are known in theart and include, but are not limited to, liposomes, lipospheres,polymers (e.g., polymer-conjugates), peptides, proteins such asantibodies (e.g., immunoconjugates, such as Antibody Drug Conjugates(ADCs) and antigen binding antibody fragments and derivatives thereof),cellular components, cyclic oligosaccharides (e.g., cyclodextrins),micelles, microparticles (e.g., microspheres), nanoparticles (e.g.,lipid nanoparticles, biodegradable nanoparticles, and core-shellnanoparticles), hydrogels, lipoprotein particles, viral sequences, viralmaterial, or lipid or liposome formulations, and combinations thereof.The delivery vehicle can be linked directly or indirectly to a targetingmoiety. In some examples, the targeting moiety is selected from among amacromolecule, a protein, a peptide, a monoclonal antibody or a fattyacid lipid.

A “subject” refers to a human or vertebrate mammal including but notlimited to a dog, cat, horse, goat and primate, e.g., monkey. Thus, theinvention can also be used to treat diseases or conditions in non-humansubjects. For instance, cancer is one of the leading causes of death incompanion animals (e.g., cats and dogs). In some embodiments, of theinvention, the subject is a human. In this disclosure, the term“subject” and “patient” is used interchangeably and has the samemeaning. It is preferred generally that a maximum dose be used, that is,the highest safe dose according to sound medical judgment.

As used herein an “effective amount” refers to a dosage of an agentsufficient to provide a medically desirable result. The effective amountwill vary with the desired outcome, the particular condition beingtreated or prevented, the age and physical condition of the subjectbeing treated, the severity of the condition, the duration of thetreatment, the nature of the concurrent or combination therapy (if any),the specific route of administration and like factors within theknowledge and expertise of the health practitioner. An “effectiveamount” can be determined empirically and in a routine manner, inrelation to the stated purpose. In the case of cancer, the effectiveamount of an agent may reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and preferably stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and preferably stop) tumor metastasis; inhibit, to someextent, tumor growth; and/or relieve to some extent one or more of thesymptoms associated with the disorder. To the extent the drug mayprevent growth and/or kill existing cancer cells, it may be cytostaticand/or cytotoxic. For cancer therapy, efficacy in vivo can, for example,be measured by assessing the duration of survival, duration ofprogression free survival (PFS), the response rates (RR), duration ofresponse, and/or quality of life.

The terms “hyperproliferative disorder”, “proliferative disease”, and“proliferative disorder”, are used interchangeably herein to pertain toan unwanted or uncontrolled cellular proliferation of excessive orabnormal cells which is undesired, such as, neoplastic or hyperplasticgrowth, whether in vitro or in vivo. In some embodiments, theproliferative disease is cancer or tumor disease (including benign orcancerous) and/or any metastases, wherever the cancer, tumor and/or themetastasis is located. In some embodiments, the proliferative disease isa benign or malignant tumor. In some embodiments, the proliferativedisease is a non-cancerous disease. In some embodiments, theproliferative disease is a hyperproliferative condition such ashyperplasias, fibrosis (especially pulmonary, but also other types offibrosis, such as renal fibrosis), angiogenesis, psoriasis,atherosclerosis and smooth muscle proliferation in the blood vessels,such as stenosis or restenosis following angioplasty.

“Cancer”, “tumor”, or “malignancy”, are used as synonymous terms andrefer to any of a number of cell types or diseases that arecharacterized by uncontrolled, abnormal proliferation of cells, theability of affected cells to spread locally or through the bloodstreamand lymphatic system to other parts of the body (metastasize) and/or anyof the characteristic structural and/or molecular features known to beassociated with these cell types or diseases. “Tumor”, as used hereinrefers to all neoplastic cell growth and proliferation, whethermalignant or benign, and all pre-cancerous and cancerous cells andtissues. A “cancerous tumor”, or “malignant cell” is understood as acell having specific structural properties, lacking differentiation andbeing capable of invasion and metastasis. A cancer that can be treatedusing a αPTHF composition provided herein includes without limitation, anon-hematologic malignancy including such as for example, lung cancer,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, headand neck cancer, gastric cancer, gastrointestinal cancer, colorectalcancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer,biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, andmelanoma; and a hematologic malignancy such as for example, a leukemia,a lymphoma and other B cell malignancies, myeloma and other plasma celldysplasias or dyscrasias. In some embodiments, the cancer is selectedfrom the group consisting of: colorectal cancer, breast cancer, gastriccancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lungcancer (e.g., non-small cell lung cancer and/or adenocarcinoma), headand neck cancer, ovarian cancer, gallbladder cancer, and basal cellcancer.

Other types of cancer and tumors that may be treated using a αPTHFcomposition are described herein or otherwise known in the art. Theterms “cancer,” “cancerous,” “cell proliferative disorder,”“proliferative disorder,” and “tumor” are not mutually exclusive asreferred to herein.

Terms such as “treating”, “treatment”, or “to treat”, refer to both (a)therapeutic measures that cure, slow down, lessen symptoms of, and/orhalt progression of a diagnosed pathologic condition or disorder and (b)prophylactic or preventative measures that prevent and/or slow thedevelopment of a targeted disease or condition. Thus, subjects in needof treatment include those already with the cancer, disorder or disease;those at risk of having the cancer or condition; and those in whom theinfection or condition is to be prevented. Subjects are identified as“having or at risk of having” cancer, an infectious disease, a disorderof the immune system, a hyperproliferative disease, or another diseaseor disorder referred to herein using well-known medical and diagnostictechniques. In certain embodiments, a subject is successfully “treated”according to the methods provided herein if the subject shows, e.g.,total, partial, or transient amelioration or elimination of a symptomassociated with the disease or condition (e.g., cancer, inflammation,and rheumatoid arthritis). In specific embodiments, the terms treating”,or “treatment”, or “to treat”, refer to the amelioration of at least onemeasurable physical parameter of a proliferative disorder, such asgrowth of a tumor, not necessarily discernible by the patient. In otherembodiments, the terms treating”, or “treatment”, or “to treat”, referto the inhibition of the progression of a proliferative disorder, eitherphysically by, e.g., stabilization of a discernible symptom,physiologically by, e.g., stabilization of a physical parameter, orboth. In other embodiments, the terms treating”, or “treatment”, or “totreat”, refer to the reduction or stabilization of tumor size, tumorcell proliferation or survival, or cancerous cell count. Treatment canbe with a α-PTHF composition, alone or in combination with an additionaltherapeutic agent.

“Subject”, “patient”, and “animal”, are used interchangeably and referto mammals such as human patients and non-human primates, as well asexperimental animals such as rabbits, rats, and mice, and other animals.Animals include all vertebrates, e.g., mammals and non-mammals, such aschickens, amphibians, and reptiles. “Mammal” as used herein refers toany member of the class Mammalia, including, without limitation, humansand nonhuman primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingrodents such as mice, rats and guinea pigs, and other members of theclass Mammalia known in the art. In a particular embodiment, the subjectis a human.

“Treatment of a proliferative disorder” is used herein to includemaintaining or decreasing tumor size, inducing tumor regression (eitherpartial or complete), inhibiting tumor growth, and/or increasing thelife span of a subject having the proliferative disorder. In oneembodiment, the proliferative disorder is a solid tumor. Such tumorsinclude, for example, lung cancer, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, head and neck cancer, gastric cancer,gastrointestinal cancer, colorectal cancer, esophageal cancer, cervicalcancer, liver cancer, kidney cancer, biliary duct cancer, gallbladdercancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer,central nervous system cancer, and melanoma. In one embodiment, theproliferative disorder is a hematologic malignancy. Such hematologicmalignancies include for example, a leukemia, a lymphoma and other Bcell malignancies, myeloma and other plasma cell dysplasias ordyscrasias.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addison's disease, alopecia areata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, inflammation andrheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, and ulcerative colitis, among others.

The term “therapeutic agent” is used herein to refer to an agent or aderivative thereof that can interact with a hyperproliferative cell suchas a cancer cell or an immune cell, thereby reducing the proliferativestatus of the cell and/or killing the cell. Examples of therapeuticagents include, but are not limited to, chemotherapeutic agents,cytotoxic agents, platinum-based agents (e.g., cisplatin, carboplatin,oxaliplatin), taxanes (e.g., TAXOL®), etoposide, alkylating agents(e.g., cyclophosphamide, ifosamide), metabolic antagonists (e.g.,tetrahydrofolate (THF), 5-fluorouracil gemcitabine, or derivativesthereof), antitumor antibiotics (e.g., mitomycin, doxorubicin),plant-derived antitumor agents (e.g., vincristine, vindesine, TAXOL®).Such agents may further include, but are not limited to, the anticanceragents trimetrexate, temozolomide, S-(4-Nitrobenzyl)-6-thioinosine(NBMPR), 6-benzyguanidine (6-BG), bis-chloronitrosourea (BCNU) andcamptothecin, or a therapeutic derivative of any thereof. Additionalexamples of therapeutic agents that may be suitable for use inaccordance with the disclosed methods include, without limitation,anti-restenosis, pro- or anti-proliferative, anti-inflammatory,anti-neoplastic, antimitotic, anti-platelet, anticoagulant, antifibrin,antithrombin, cytostatic, antibiotic and other anti-infective agents,anti-enzymatic, anti-metabolic, angiogenic, cytoprotective, angiotensinconverting enzyme (ACE) inhibiting, angiotensin II receptor antagonizingand/or cardioprotective agents. “Therapeutic agents” also refer tosalts, acids, and free based forms of the above agents.

As used herein, the term “chemotherapeutic agent” when used in relationto cancer therapy, refers to any agent that results in the death ofcancer cells or inhibits the growth or spread of cancer cells. Examplesof such chemotherapeutic agents include alkylating agents, antibiotics,antimetabolites, plant-derived agents, and hormones. In someembodiments, the disclosed polyglutamated alpha tetrahydrofolatecompositions are used in combination with a chemotherapeutic agent. Insome embodiments, the chemotherapeutic agent is 5-fluorouracil. In someembodiments, the chemotherapeutic agent is cisplatin. In someembodiments, the chemotherapeutic agent is carboplatin. In someembodiments, the chemotherapeutic agent is oxaliplatin. In otherembodiments, the chemotherapeutic agent is gemcitabine. In otherembodiments, the chemotherapeutic agent is doxorubicin. In particularembodiments, the chemotherapeutic agent is a pyrimidine analog (e.g., afluorpyrimidine such as 5-fluorouracil (5-FU)).

The term “antimetabolite” is used herein to refer to a therapeutic agentthat inhibits the utilization of a metabolite or a prodrug thereof.Examples of antimetabolites include 5-FU and 5-FU metabolites and/orprodrugs such as 5-FUMP, 5-FUDP, 5-FdUMP, capecitabine, tegafur5-fluorodeoxyuridine monophosphate; and cytarabine and cytarabineprodrugs such as nelarabine, 5-azacytidine, gemcitabine, mercaptopurine,thioguanine, azathioprine, adenosine, pentostatin,erythrohydroxynonyladenine, and cladribine. Anti-metabolites useful forpracticing the disclosed methods include nucleoside analogs, including apurine or pyrimidine analogs. In some embodiments, the polyglutamatedalpha tetrahydrofolate compositions are used in combination with anantimetabolite selected from the group consisting of: afluoropyrimidine, 5-fluorouracil,5-fluoro-1-(oxolan-2-yl)pyrimidine-2,4-dione, 5-fluoro-2′-deoxycytidine,cytarabine, gemcitabine, troxacitabine, decitabine, Azacytidine,pseudoisocytidine, Zebularine, Ancitabine, Fazarabine, 6-azacytidine,capecitabine, N4-octadecyl-cytarabine, elaidic acid cytarabine,fludarabine, cladribine, clofarabine, nelarabine, forodesine, andpentostatin, or a derivative thereof. In one example, the nucleosideanalog is a substrate for a nucleoside deaminase that is adenosinedeaminase or cytidine deaminase. In some examples, the nucleoside analogis selected from among fludarabine, cytarabine, gemcitabine, decitabineand azacytidine or derivatives thereof. In some examples, the nucleosideanalog is selected from N3-alkylated analogues of 5-fluorouracil,5-fluorouracil derivatives with 1,4-oxaheteroepane moieties,5-fluorouracil and nucleoside analogues, cis- andtrans-5-fluoro-5,6-dihydro-6-alkoxyuracil, cyclopentane 5-fluorouracilanalogues, A-OT-fluorouracil,N4-trimethoxybenzoyl-5′-deoxy-5-fluoro-cytidine and5′-deoxy-5-fluorouridine, 1-hexylcarbamoyl-5-fluorouracil, B-3839,uracil-1-(2-tetrahydrofuryl)-5-fluorouracil,1-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)-5-fluorouracil,doxifluridine, 5′-deoxy-5-fluorouridine,1-acetyl-3-O-toluoyl-5-fluorouracil,5-fluorouracil-m-formylbenzene-sulfonate (JP 55059173),N′-(2-furanidyl)-5-fluorouracil (JP 53149985) and1-(2-tetrahydrofuryl)-5-fluorouracil or derivatives thereof. Inparticular embodiments, the antimetabolite is a pyrimidine analog or apyrimidine analog prodrug (e.g., a fluorpyrimidine). In certainembodiments, the antimetabolite is 5-fluorouracil.

As used herein, a “taxane” is an anti-cancer agent that interferes withor disrupts microtubule stability, formation and/or function. Taxaneagents include paclitaxel and docetaxel as well as derivatives thereof,wherein the derivatives function against microtubules by the same modeof action as the taxane from which they are derived. In certainembodiments, the taxane is paclitaxel or docetaxel, or apharmaceutically acceptable salt, acid, or derivative of paclitaxel ordocetaxel. In certain embodiments, the taxane is paclitaxel (TAXOL®),docetaxel (TAXOTERE®), albumin-bound paclitaxel (nab-paclitaxel;ABRAXANE®), DHA-paclitaxel, or PG-paclitaxel.

The term “pharmaceutically-acceptable carrier” refers to an ingredientin a pharmaceutical formulation, other than an active ingredient, whichis nontoxic to a subject. A pharmaceutically acceptable carrierincludes, but is not limited to, a buffer, carrier, excipient,stabilizer, diluent, or preservative. Pharmaceutically-acceptablecarriers can include for example, one or more compatible solid or liquidfiller, diluents or encapsulating substances which are suitable foradministration to a human or other subject.

This disclosure generally relates to polyglutamated alphatetrahydrofolate (αPTHF) compositions and methods of making and usingthe compositions to treat diseases including hyperproliferative diseasessuch as cancer, disorders of the immune system such as rheumatoidarthritis, and infectious disease such as HIV and malaria. The gammapolyglutamated compositions also have uses in combination therapy withone or more therapeutic agents such as a chemotherapy drug (e.g.,5-fluorouracil) to enhance the effectiveness of the therapeutic agent(s)or as a “chemoprotectant” (e.g., in combination with an antifolate suchas methotrexate) to reduce toxic side effects associated with thetherapeutic agent(s).

In some embodiments, the disclosure provides:

-   -   [1] a composition comprising a polyglutamated alpha        tetrahydrofolate.    -   [2] the composition of [1], wherein the polyglutamated alpha        tetrahydrofolate is selected from the group consisting of:        -   (a) polyglutamated 5-formyl-THF (e.g., polyglutamated            [6S]-5-formyl-THF);        -   (b) polyglutamated 10-formyl-THF (e.g., polyglutamated            [6R]-10-formyl-THF);        -   (c) polyglutamated 5,10-methenyl-THF (e.g., polyglutamated            [6R]-5,10-methenyl-THF);        -   (d) polyglutamated 5-methyl-THF (e.g., polyglutamated            [6S]-5-methyl-THF);        -   (e) polyglutamated tetrahydrofolate (e.g., polyglutamated            [6S]-Tetrahydrofolate THF);        -   (f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated            [6R]-5,10-methylene-THF); and        -   (g) polyglutamated 5-formimino-THF (e.g., polyglutamated            [6S]-5-formimino-THF).    -   [3] the composition of [1] or [2], wherein the polyglutamated        alpha tetrahydrofolate contains 4, 5, 2-10, 4-6, or more than 5,        glutamyl groups having alpha carboxyl group linkages.    -   [4] the composition according to any of [1]-[3], wherein the        polyglutamated alpha tetrahydrofolate is tetraglutamated alpha        tetrahydrofolate (e.g., [6R]-5,10-methenyl-THF,        [6S]-5-formyl-THF and/or [6R]-10-formyl-THF).    -   [5] the composition according to any of [1]-[3], wherein the        polyglutamated alpha tetrahydrofolate is pentaglutamated alpha        tetrahydrofolate (e.g., [6R]-5,10-methenyl-THF,        [6S]-5-formyl-THF and/or [6R]-10-formyl-THF).    -   [6] the composition according to any of [1]-[3], wherein the        polyglutamated alpha tetrahydrofolate is hexaglutamated alpha        tetrahydrofolate (e.g., [6R]-5,10-methenyl-THF,        [6S]-5-formyl-THF and/or [6R]-10-formyl-THF).    -   [7] the composition according to any of [1] to [6], wherein        -   (a) two or more glutamyl groups have an alpha carboxyl group            linkage,        -   (b) each of the glutamyl groups other than the glutamyl            group of tetrahydrofolate has an alpha carboxyl group            linkage; or        -   (c) two or more glutamyl groups have a gamma carboxyl group            linkage.    -   [8] the composition according to any of [1] to [6], wherein        -   (a) each of the glutamyl groups other than the C-terminal            glutamyl group or groups and the glutamyl group of            tetrahydrofolate has an alpha carboxyl group linkage; or        -   (b) each of the glutamyl groups other than the C-terminal            glutamyl group or groups has an alpha carboxyl group            linkage.    -   [9] the composition according to any of [1]-[8], wherein at        least one glutamyl group has both an alpha carboxyl group        linkage and a gamma carboxyl group linkage;    -   [10] the composition according to any of [1]-[9], wherein:        -   (a) at least 2 of the glutamyl groups of the alpha            polyglutamated tetrahydrofolate are in the L-form,        -   (b) each of the glutamyl groups of the alpha polyglutamated            tetrahydrofolate is in the L-form,        -   (c) at least 1 of the glutamyl groups of the alpha            polyglutamated tetrahydrofolate is in the D-form,        -   (d) each of the glutamyl groups of the alpha polyglutamated            tetrahydrofolate other than the glutamyl group of            tetrahydrofolate is in the D-form, or        -   (e) at least 2 of the glutamyl groups of the alpha            polyglutamated tetrahydrofolate are in the L-form and at            least 1 of the glutamyl groups is in the D-form;    -   [11] the composition according to any of [1]-[10], wherein the        polyglutamate is linear;    -   [12] the composition according to any of [1]-[10], wherein the        polyglutamate is branched;    -   [13] a liposomal composition comprising the alpha polyglutamated        tetrahydrofolate according to any of [1]-[12] (Lp-αPTHF);    -   [14] the LαPP composition according to [13], wherein the alpha        polyglutamated tetrahydrofolate comprises glutamyl groups in the        L-form having alpha carboxyl group linkages;    -   [15] the Lp-αPTHF composition according to [13] or [14], wherein        each of the glutamyl groups of the alpha polyglutamated        tetrahydrofolate is in the L-form;    -   [16] the Lp-αPTHF composition of [13] or [14], wherein at least        one of the glutamyl groups of the alpha polyglutamated        tetrahydrofolate is in the D-form;    -   [17] the Lp-αPTHF composition according to any of [13]-[16],        wherein the liposome comprises an alpha polyglutamated        tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 5,        glutamyl groups;    -   [18] the Lp-αPTHF composition according to any of [13]-[17],        wherein at least one of the glutamyl groups of the alpha        polyglutamated tetrahydrofolate has a gamma carboxyl group        linkage;    -   [19] the composition according to any of [13]-[18], wherein at        least one glutamyl group has both an alpha carboxyl group        linkage and a gamma carboxyl group linkage;    -   [20] the composition according to any of [13]-[19], which        contains 2, 3, 4, 5, 2-10, 4-6, or more than 5, glutamyl groups        that have both an alpha carboxyl group linkage and a gamma        carboxyl group linkage;    -   [21] the Lp-αPTHF composition according to any of [13]-[20],        wherein the liposome comprises an alpha polyglutamated        tetrahydrofolate containing alpha tetraglutamated        tetrahydrofolate, alpha pentaglutamated tetrahydrofolate, or        alpha hexaglutamated tetrahydrofolate;    -   [22] the Lp-αPTHF composition according to any of [13]-[21],        wherein the polyglutamate is linear or branched;    -   [23] the Lp-αPTHF composition according to any of [13]-[22],        wherein the liposome is pegylated (PαLp-αPTHF);    -   [24] the Lp-αPTHF composition according to any of [13]-[23],        wherein the liposomes comprise at least 1% weight by weight        (w/w) of the alpha polyglutamated tetrahydrofolate or wherein        during the process of preparing the Lp-αPTHF, at least 1% of the        starting material of alpha polyglutamated THF is encapsulated        (entrapped) in the αPTHF;    -   [25] the Lp-αPTHF composition according to any of [13]-[24],        wherein the liposome has a diameter in the range of 20 nm to 500        nm or 20 nm to 200 nm;    -   [26] the Lp-αPTHF composition according to any of [13]-[25],        wherein the liposome has a diameter in the range of 80 nm to 120        nm;    -   [27] the Lp-αPTHF composition according to any of [13]-[26],        wherein the liposome is formed from liposomal components;    -   [28] the Lp-αPTHF composition according to [27], wherein the        liposomal components comprise at least one of an anionic lipid        and a neutral lipid;    -   [29] the Lp-αPTHF composition according to [27] or [28], wherein        the liposomal components comprise at least one selected from the        group consisting of: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC;        HSPC-PEG; cholesterol; cholesterol-PEG; and        cholesterol-maleimide;    -   [30] the Lp-αPTHF composition according to any of [27]-[29],        wherein the liposomal components comprise at least one selected        from the group consisting of: DSPE; DSPE-PEG; DSPE-PEG-FITC;        DSPE-PEG-maleimide; cholesterol; and HSPC;    -   [31] the Lp-αPTHF composition according to any of [27]-[30],        wherein one or more liposomal components further comprises a        steric stabilizer;    -   [32] the Lp-αPTHF composition according to [31], wherein the        steric stabilizer is at least one selected from the group        consisting of polyethylene glycol (PEG); poly-L-lysine (PLL);        monosialoganglioside (GM1); poly(vinyl pyrrolidone) (PVP);        poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline);        poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol;        poly[N-(2-hydroxypropyl) methacrylamide]; amphiphilic        poly-N-vinylpyrrolidones; L-amino-acid-based polymer;        oligoglycerol, copolymer containing polyethylene glycol and        polypropylene oxide, Poloxamer 188, and polyvinyl alcohol;    -   [33] the Lp-αPTHF composition according to [32], wherein the        steric stabilizer is PEG and the PEG has a number average        molecular weight (Mn) of 200 to 5000 daltons;    -   [34] the Lp-αPTHF composition according to any of [13]-[33],        wherein the liposome is anionic or neutral;    -   [35] the Lp-αPTHF composition according to any of [13]-[33],        wherein the liposome has a zeta potential that is less than or        equal to zero;    -   [36] the Lp-αPTHF composition according to any of [13]-[33],        wherein the liposome has a zeta potential that is between 0 to        −150 mV;    -   [37] the Lp-αPTHF composition according to any of [13]-[33],        wherein the liposome has a zeta potential that is between −30 to        −50 mV;    -   [38] the Lp-αPTHF composition according to any of [13]-[33],        wherein the liposome is cationic;    -   [39] the Lp-αPTHF composition according to any of [13]-[38],        wherein the liposome has an interior space comprising the alpha        polyglutamated tetrahydrofolate and an aqueous pharmaceutically        acceptable carrier;    -   [40] the Lp-αPTHF composition of [39], wherein the        pharmaceutically acceptable carrier comprises a tonicity agent        such as dextrose, mannitol, glycerine, potassium chloride,        sodium chloride, at a concentration of greater than 1%;    -   [41] the Lp-αPTHF composition of [39], wherein the aqueous        pharmaceutically acceptable carrier is trehalose;    -   [42] the Lp-αPTHF composition of [41], wherein the        pharmaceutically acceptable carrier comprises 5% to 20% weight        of trehalose;    -   [43] the Lp-αPTHF composition according to any of [39]-[42],        wherein the pharmaceutically acceptable carrier comprises 1% to        15 weight of dextrose;    -   [44] the Lp-αPTHF composition according to any of [39]-[43],        wherein the interior space of the liposome comprises 5% dextrose        suspended in an HEPES buffered solution;    -   [45] the Lp-αPTHF composition according to any of [39]-[44],        wherein the pharmaceutically acceptable carrier comprises a        buffer such as HEPES Buffered Saline (HBS) or similar, at a        concentration of between 1 to 200 mM and a pH of between 2 to 8;    -   [46] the Lp-αPTHF composition according to any of [39]-[45],        wherein the pharmaceutically acceptable carrier comprises a        total concentration of sodium acetate and calcium acetate of        between 50 mM to 500 mM;    -   [47] the Lp-αPTHF composition according to any of [13]-[46],        wherein the interior space of the liposome has a pH of 5-8 or a        pH of 6-7, or any range therein between;    -   [48] the Lp-αPTHF composition according to any of [13]-[47],        wherein the liposome comprises less than 500,000 or less than        200,000 molecules of the alpha polyglutamated tetrahydrofolate;    -   [49] the Lp-αPTHF composition according to any of [13]-[48],        wherein the liposome comprises between 10 to 100,000 molecules        of the alpha polyglutamated tetrahydrofolate, or any range        therein between;    -   [50] the Lp-αPTHF composition according to any of [13]-[49],        which further comprises a targeting moiety and wherein the        targeting moiety has a specific affinity for a surface antigen        on a target cell of interest;    -   [51] the Lp-αPTHF composition according to [50], wherein the        targeting moiety is attached to one or both of a PEG and the        exterior of the liposome, optionally wherein targeting moiety is        attached to one or both of the PEG and the exterior of the        liposome by a covalent bond;    -   [52] the Lp-αPTHF composition of [50] or [51], wherein the        targeting moiety is a polypeptide;    -   [53] the Lp-αPTHF composition according to any of [50]-[52],        wherein the targeting moiety is an antibody or an antigen        binding fragment of an antibody;    -   [54] the Lp-αPTHF composition according to any of [50]-[53],        wherein the targeting moiety binds the surface antigen with an        equilibrium dissociation constant (Kd) in a range of 0.5×10⁻¹⁰        to 10×10⁻⁶ as determined using BIACORE® analysis;    -   [55] the Lp-αPTHF composition according to any of [50]-[55],        wherein the targeting moiety specifically binds one or more        folate receptors selected from the group consisting of: folate        receptor alpha (FR-α), folate receptor beta (FR-β), and folate        receptor delta (FR-δ);    -   [56] the Lp-αPTHF composition according to any of [50]-[56],        wherein the targeting moiety comprises one or more selected from        the group consisting of: an antibody, a humanized antibody, an        antigen binding fragment of an antibody, a single chain        antibody, a single-domain antibody, a bi-specific antibody, a        synthetic antibody, a pegylated antibody, and a multimeric        antibody;    -   [57] the Lp-αPTHF composition according to any of [50]-[56],        wherein each pegylated liposome comprises from 1 to 1000 or        30-200 targeting moieties;    -   [58] the Lp-αPTHF composition according to any of [39]-[57],        further comprising one or more of an immunostimulatory agent, a        detectable marker and a maleimide, wherein the immunostimulatory        agent, the detectable marker or the maleimide is attached to        said PEG or the exterior of the liposome;    -   [59] the Lp-αPTHF composition of [58], wherein the        immunostimulating agent is at least one selected from the group        consisting of: a protein immunostimulating agent; a nucleic acid        immunostimulating agent; a chemical immunostimulating agent; a        hapten; and an adjuvant;    -   [60] the Lp-αPTHF composition of [58] or [59], wherein the        immunostimulating agent is at least one selected from the group        consisting of: a fluorescein; a fluorescein isothiocyanate        (FITC); a DNP; a beta glucan; a beta-1,3-glucan; a        beta-1,6-glucan; a resolvin (e.g., a Resolvin D such as        D_(n-6DPA) or D_(n-3DPA), a Resolvin E, or a T series resolvin);        and a Toll-like receptor (TLR) modulating agent such as, an        oxidized low-density lipoprotein (e.g., OXPAC, PGPC), and an        eritoran lipid (e.g., E556);    -   [61] the Lp-αPTHF composition according to any of [58]-[60],        wherein the immunostimulatory agent and the detectable marker is        the same;    -   [62] the Lp-αPTHF composition according to any of [58]-[61],        further comprising a hapten;    -   [63] the Lp-αPTHF composition of [62], wherein the hapten        comprises one or more of fluorescein or Beta 1, 6-glucan;    -   [64] the Lp-αPTHF composition according to any of [13]-[63],        which further comprises in the interior space, the exterior        space, or both the interior space at least one cryoprotectant        selected from the group consisting of mannitol; trehalose;        sorbitol; and sucroseat least one cryoprotectant selected from        the group consisting of mannitol; trehalose; sorbitol; and        sucrose;    -   [65] a targeted composition comprising the composition according        to any of [1]-[64];    -   [66] An non-targeted composition comprising the composition        according to any of [1]-[49];    -   [67] the Lp-αPTHF composition according to any of [13]-[66],        which further comprises carboplatin and/or pembroluzumab;    -   [68] a pharmaceutical composition comprising the liposomal alpha        polyglutamated tetrahydrofolate composition according to any of        [13]-[67];    -   [69] a pharmaceutical composition comprising alpha        polyglutamated tetrahydrofolate composition according to any of        [1]-[8];    -   [70] the composition of any of [1]-[69], for use in the        treatment of disease;    -   [71] use of the composition of any of [1]-[70], in the        manufacture of a medicament for the treatment of disease;    -   [72] use of the composition of any of [1]-[70], in the        manufacture of a medicament for the treatment of disease and/or        for use in combination therapy with one or more therapeutic        agents such as a chemotherapeutic drug (e.g., 5-fluorouracil) to        enhance the effectiveness of the therapeutic agent(s) or as a        “chemoprotectant” (e.g., in combination with an antifolate such        as methotrexate) to reduce a toxic side effect associated with        the therapeutic agent(s);    -   [73] a method for treating or preventing disease in a subject        needing such treatment or prevention, the method comprising        administering the composition of any of    -   [1]-[69] to the subject;    -   [74] a method for treating or preventing disease in a subject        needing such treatment or prevention, the method comprising        administering the liposomal polyglutamated alpha        tetrahydrofolate composition of any of [13]-[69] to the subject;    -   [75] a method of killing a hyperproliferative cell that        comprises contacting a hyperproliferative cell with the        composition of any of [1]-[69];    -   [76] a method of killing a hyperproliferative cell that        comprises contacting a hyperproliferative cell with the        liposomal polyglutamated alpha tetrahydrofolate composition of        any of [13]-[69];    -   [77] the method of [75] or [76], wherein the hyperproliferative        cell is a cancer cell, a mammalian cell, and/or a human cell;    -   [78] a method for treating cancer that comprises administering        an effective amount of the composition of any of [1]-[69] to a        subject having or at risk of having cancer;    -   [79] a method for treating cancer that comprises administering        an effective amount of the liposomal polyglutamated alpha        tetrahydrofolate composition of any of [13]-[68] to a subject        having or at risk of having cancer;    -   [80] the method of [78] or [79], wherein the method treats or        prevents cancer and wherein the cancer is selected from the        group consisting of: a non-hematologic malignancy including such        as for example, lung cancer, pancreatic cancer, breast cancer,        ovarian cancer, prostate cancer, head and neck cancer, gastric        cancer, gastrointestinal cancer, colorectal cancer, esophageal        cancer, cervical cancer, liver cancer, kidney cancer, biliary        duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,        osteosarcoma), brain cancer, central nervous system cancer, and        melanoma; and a hematologic malignancy such as for example, a        leukemia, a lymphoma and other B cell malignancies, myeloma and        other plasma cell dyscrasias;    -   [81] the method of [78] or [79], wherein the method treats or        prevents cancer and wherein the cancer is a member selected from        the group consisting of: lung cancer, breast cancer, colon        cancer, pancreatic cancer, gastric cancer, bladder cancer, head        and neck cancer, ovarian cancer, and cervical cancer;    -   [82] the method of [78] or [79], wherein the method treats or        prevents cancer and wherein the cancer is a member selected from        the group consisting of: colorectal cancer, lung cancer, breast        cancer, head and neck cancer, and pancreatic cancer;    -   [83] the method of [78] or [79], wherein the method treats or        prevents cancer and wherein the cancer is selected from the        group consisting of: colorectal cancer, breast cancer, ovarian        cancer, lung cancer, head and neck cancer, pancreatic cancer,        gastric cancer, and mesothelioma;    -   [84] a method for treating cancer that comprises administering        an effective amount of the Lp-αPTHF composition of any of        [50]-[66] to a subject having or at risk of having a cancer cell        that expresses on its surface a folate receptor bound by the        targeting moiety;    -   [85] a maintenance therapy for subjects that are undergoing or        have undergone cancer therapy that comprise administering an        effective amount of the composition of any of [1]-[69] to a        subject that is undergoing or has undergone cancer therapy;    -   [86] a maintenance therapy for subjects that are undergoing or        have undergone cancer therapy that comprise administering an        effective amount of the liposomal polyglutamated alpha        tetrahydrofolate composition of any of [13]-[69] to a subject        that is undergoing or has undergone cancer therapy;    -   [87] a method for treating a disorder of the immune system that        comprises administering an effective amount of the composition        of any of [1]-[69] to a subject having or at risk of having a        disorder of the immune system, optionally wherein the disorder        of the immune system is selected from: inflammation (e.g., acute        and chronic), systemic inflammation, rheumatoid arthritis,        inflammatory bowel disease (IBD), Crohn disease,        dermatomyositis/polymyositis, systemic lupus erythematosus, and        Takayasu, and psoriasis;    -   [88] a method for treating a disorder of the immune system that        comprises administering an effective amount of the liposomal        polyglutamated alpha tetrahydrofolate composition of any of        [13]-[69] to a subject having or at risk of having a disorder of        the immune system, optionally wherein the disorder of the immune        system is selected from: inflammation (e.g., acute and chronic),        systemic inflammation, rheumatoid arthritis, inflammatory bowel        disease (IBD), Crohn disease, dermatomyositis/polymyositis,        systemic lupus erythematosus, and Takayasu, and psoriasis;    -   [89] a method for treating:        -   (a) leukopenia that comprises administering an effective            amount of the composition according to any of [1]-[69] to a            subject having or at risk of having leukopenia;        -   (b) an infectious disease that comprises administering an            effective amount of the composition according to any of            [1]-[69] to a subject having or at risk of having an            infectious disease;        -   (c) cardiovascular disease or metabolic disease that            comprises administering an effective amount of the            composition according to any of [1]-[69] to a subject having            or at risk of having an infectious disease, cardiovascular            disease, or another disease, wherein the disease is a member            selected from: atherosclerosis, cardiovascular disease            (CVD), coronary artery disease, myocardial infarction,            stroke, metabolic syndrome, a gestational trophoblastic            disease, and ectopic pregnancy;        -   (d) an autoimmune disease, that comprises administering an            effective amount of the composition according to any of            [1]-[69] to a subject having or at risk of having an            autoimmune disease;        -   (e) rheumatoid arthritis, that comprises administering an            effective amount of the composition according to any of            [1]-[69] to a subject having or at risk of having rheumatoid            arthritis;        -   (f) an inflammatory condition that comprises administering            an effective amount of the composition according to any of            [1]-[69] to a subject having or at risk of having            inflammation, optionally wherein the inflammation is acute,            chronic, and/or systemic inflammation; or        -   (g) a skin condition that comprises administering an            effective amount of the composition according to any of            [1]-[69] to a subject having or at risk of having a skin            condition, optionally wherein the skin condition is            psoriasis;    -   [90] a method for treating an infectious disease that comprises        administering an effective amount of the liposomal alpha        polyglutamated tetrahydrofolate composition of any of [13]-[69]        to a subject having or at risk of having an infectious disease;    -   [91] a method of delivering alpha polyglutamated        tetrahydrofolate to a tumor expressing a folate receptor on its        surface, the method comprising: administering the Lp-αPTHF        composition of any of [1]-[69] to a subject having the tumor in        an amount to deliver a therapeutically effective dose of the        alpha polyglutamated tetrahydrofolate to the tumor;    -   [92] a method of preparing an alpha polyglutamated        tetrahydrofolate composition comprising the liposomal alpha        polyglutamated tetrahydrofolate composition of any of [13]-[69],        the method comprising: forming a mixture comprising: liposomal        components and alpha polyglutamated antifolate in solution;        homogenizing the mixture to form liposomes in the solution; and        processing the mixture to form liposomes containing alpha        polyglutamated tetrahydrofolate;    -   [93] a method of preparing an alpha polyglutamated        tetrahydrofolate composition comprising the liposomal alpha        polyglutamated tetrahydrofolate composition of any of [13]-[69],        the method comprising: forming a mixture comprising: liposomal        components and alpha polyglutamated tetrahydrofolate in        solution; and processing the mixture to form liposomes        containing alpha polyglutamated tetrahydrofolate,    -   [94] the method of [93], wherein the processing the mixture        comprises homogenizing the mixture to form liposomes in the        solution,    -   [95] a method of preparing the composition of any of [51]-[70]        comprising the steps of: forming a mixture comprising: liposomal        components and alpha polyglutamated tetrahydrofolate in a        solution; homogenizing the mixture to form liposomes in the        solution; processing the mixture to form liposomes entrapping        and/or encapsulating alpha polyglutamated tetrahydrofolate; and        providing a targeting moiety on a surface of the liposomes, the        targeting moiety having specific affinity for at least one of        folate receptor alpha (FR-α), folate receptor beta (FR-β) and        folate receptor delta (FR-δ);    -   [96] a method of preparing the composition of any of [51]-[70],        comprising the steps of: forming a mixture comprising: liposomal        components and alpha polyglutamated tetrahydrofolate in a        solution; processing the mixture to form liposomes entrapping        and/or encapsulating alpha polyglutamated tetrahydrofolate; and        providing a targeting moiety on a surface of the liposomes, the        targeting moiety having specific affinity for at least one of        folate receptor alpha (FR-α), folate receptor beta (FR-β) and        folate receptor delta (FR-δ);    -   [97] the method of [96], wherein the processing step comprises        homogenizing the mixture to form liposomes in the solution,    -   [98] the method according to [93], wherein the processing step        includes one or more steps of: thin film hydration, extrusion,        in-line mixing, ethanol injection technique,        freezing-and-thawing technique, reverse-phase evaporation,        dynamic high pressure microfluidization, microfluidic mixing,        double emulsion, freeze-dried double emulsion, 3D printing,        membrane contactor method, and stirring; and/or    -   [99] the method according to any of [96] to [98], wherein said        processing step includes one or more steps of modifying the size        of the liposomes by one or more of steps of extrusion,        high-pressure microfluidization, and/or sonication; and/or    -   [100] the method of any of [92] to [99], wherein at least 1% of        the starting material of alpha polyglutamated tetrahydrofolate        is encapsulated or entrapped in the liposomes.

II. Polyglutamated alpha tetrahydrofolate (αPTHF)

The disclosure generally relates to polyglutamated alphatetrahydrofolate (αPTHF) compositions. The αPTHF compositions compriseat least one glutamyl group having an alpha carboxyl group linkage.These compositions are structurally distinct from the L-gammapolyglutamated forms of tetrahydrofolate (that are produced by theenzyme folylpoly-gamma- In some embodiments, the αPTHF compositioncontains 2-20, 2-15, 2-10, 2-5, 2-6, or more than 5, glutamyl groups(including the glutamyl group in tetrahydrofolate). In some embodiments,each of the glutamyl groups in the αPTHF other than the glutamyl groupof tetrahydrofolate, have an alpha linkage. In some embodiments, each ofthe glutamyl groups in the αPTHF other than the C-terminal glutamylgroup or groups and the glutamyl group of tetrahydrofolate, have analpha linkage. In some embodiments, each of the glutamyl groups in theαPTHF other than the C-terminal glutamyl group or groups have an alphalinkage. In some embodiments, 2 or more of the glutamyl groups in theαPTHF have a gamma linkage. In some embodiments, at least one glutamylgroup of the alpha polyglutamated tetrahydrofolate has both an alphacarboxyl group linkage and a gamma carboxyl group linkage. In someembodiments, each of the glutamyl groups in the αPTHF is in the L-form.In some embodiments, each of the glutamyl groups in the αPTHF other thanthe glutamyl group of tetrahydrofolate, is in the D-form. In someembodiments, the αPTHF comprises two or more glutamyl groups in theL-form and one or more glutamyl groups in the D-form. In someembodiments, the polyglutamate chain of the αPTHF is linear (notbranched). In some embodiments, the polyglutamate chain of the αPTHF isbranched.

In some embodiments, the alpha polyglutamated tetrahydrofolate isdiglutamated. That is, the alpha polyglutamated tetrahydrofolatecontains 1 additional glutamyl group in addition to the glutamyl groupof tetrahydrofolate (αTHF-PG₁), and the additional glutamyl group islinked to the glutamyl group in tetrahydrofolate through an alphalinkage. In some embodiments, each of the glutamyl groups of the alphadiglutamated tetrahydrofolate is in the L-form. In other embodiments,the alpha diglutamated THF comprises a glutamyl group in the D-form.

In some embodiments, the alpha polyglutamated tetrahydrofolate istriglutamated. That is, the alpha polyglutamated tetrahydrofolatecontains 2 additional glutamyl groups in addition to the glutamyl groupof tetrahydrofolate (αTHF-PG₂). In some embodiments, each of the 2additional glutamyl groups have an alpha linkage. In other embodiments,one of the 2 additional glutamyl groups have an alpha linkage and theother glutamyl group has a gamma linkage. In some embodiments, one ofthe 2 additional glutamyl groups has an alpha linkage. In someembodiments, one of the 2 additional glutamyl groups has a gammalinkage. In some embodiments, two of the three glutamyl groups have analpha linkage. In other embodiments, one of the three glutamyl groupshas an alpha linkage and another glutamyl group has a gamma linkage. Insome embodiments, one glutamyl group has both an alpha linkage and agamma linkage. In some embodiments, each of the glutamyl groups of thealpha triglutamated tetrahydrofolate is in the L-form. In otherembodiments, the alpha triglutamated THF comprises a glutamyl group inthe D-form. In further embodiments, each of the glutamyl groups of thealpha triglutamated tetrahydrofolate other than the glutamyl group oftetrahydrofolate, is in the D-form. In additional embodiments, thetriglutamated THF comprises a glutamyl group in the D-form and two ormore glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate istetraglutamated and thus contains 3 additional glutamyl groups inaddition to the glutamyl group in tetrahydrofolate (αTHF-PG₃). In someembodiments, each of the 3 additional glutamyl groups have an alphalinkage. In other embodiments, 1 or 2 of the 3 additional glutamylgroups have an alpha linkage and the remaining 2 or 1 glutamyl groups,respectively, have a gamma linkage. In some embodiments, 2 of the 3additional glutamyl groups have an alpha linkage. In other embodiments,one of the 3 additional glutamyl groups has an alpha linkage and anotheradditional glutamyl group has a gamma linkage. In other embodiments, oneof the 3 additional glutamyl groups has an alpha linkage and a gammalinkage. In other embodiments, three of the four glutamyl groups have analpha linkage. In some embodiments, at least one glutamyl group has bothan alpha linkage and a gamma linkage. In some embodiments, the alphatetraglutamated THF comprises two or more glutamyl groups in the L-form.In further embodiments, each of the glutamyl groups of the alphatetraglutamated tetrahydrofolate is in the L-form. In other embodiments,the alpha tetraglutamated THF comprises a glutamyl group in the D-form.In further embodiments, each of the glutamyl groups of the alphatetraglutamated tetrahydrofolate other than the glutamyl group oftetrahydrofolate, is in the D-form. In additional embodiments, thetetraglutamated THF comprises a glutamyl group in the D-form and two ormore glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate ispentaglutamated (αTHF-PG₄) and contains a chain of 4 additional glutamylgroups attached to the glutamyl group of tetrahydrofolate. In someembodiments, each of the 4 additional glutamyl groups in the chain havean alpha linkage. In some embodiments, each of the 4 additional glutamylgroups in the chain other than the C-terminal glutamyl group or groupshave an alpha linkage. In other embodiments, 1, 2, or 3, of the 4additional glutamyl groups have an alpha linkage and the remaining 3, 2,or 1, glutamyl groups, respectively, are linked to a glutamyl group ofthe molecule through a gamma linkage. In other embodiments, 1 or 2 ofthe 4 additional glutamyl groups have an alpha linkage and the remainingnon-C-terminal glutamyl groups are linked to a glutamyl group of themolecule through a gamma linkage. In some embodiments, at least oneadditional glutamyl group has both an alpha linkage and a gamma linkage.In some embodiments, at least one of the 5 glutamyl groups has both analpha linkage and a gamma linkage. In some embodiments, each of the 5glutamyl groups in the chain other than the C-terminal glutamyl group orgroups have an alpha linkage. In some embodiments, the alphapentaglutamated THF comprises two or more glutamyl groups in the L-form.In further embodiments, each of the glutamyl groups of the alphapentaglutamated tetrahydrofolate is in the L-form. In other embodiments,the alpha pentaglutamated THF comprises a glutamyl group in the D-form.In further embodiments, each of the glutamyl groups of the alphapentaglutamated tetrahydrofolate other than the glutamyl group oftetrahydrofolate, is in the D-form. In additional embodiments, thepentaglutamated THF comprises a glutamyl group in the D-form and two ormore glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate ishexaglutamated (αTHF-PG₅) and contains a chain of 5 additional glutamylgroups attached to the glutamyl group of tetrahydrofolate. In someembodiments, each of the 5 additional glutamyl groups in the chain havean alpha linkage. In some embodiments, each of the 5 additional glutamylgroups in the chain other than the C-terminal glutamyl group or groupshave an alpha linkage. In some embodiments, 4 of the 5 additionalglutamyl groups in the chain have an alpha linkage. In otherembodiments, 1, 2, 3, or 4, of the 5 additional glutamyl groups arelinked to a glutamyl group of the molecule through an alpha linkage andthe remaining 4, 3, 2, or 1, glutamyl groups, respectively, are linkedto a glutamyl group of the molecule through a gamma linkage. In otherembodiments, 1, 2, 3, or 4 of the 5 additional glutamyl groups have analpha linkage and the remaining non-C-terminal glutamyl groups arelinked to a glutamyl group of the molecule through a gamma linkage. Insome embodiments, at least one additional glutamyl group has both analpha linkage and a gamma linkage. In some embodiments, at least one ofthe 6 glutamyl groups has both an alpha linkage and a gamma linkage. Insome embodiments, each of the 6 glutamyl groups other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 5 of the 6 glutamyl groups have an alpha linkage. In someembodiments, the alpha hexaglutamated THF comprises two or more glutamylgroups in the L-form. In further embodiments, each of the glutamylgroups of the alpha hexaglutamated tetrahydrofolate is in the L-form. Inother embodiments, the alpha hexaglutamated THF comprises a glutamylgroup in the D-form. In further embodiments, each of the glutamyl groupsof the alpha hexaglutamated tetrahydrofolate other than the glutamylgroup of tetrahydrofolate, is in the D-form. In additional embodiments,the hexaglutamated THF comprises a glutamyl group in the D-form and twoor more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate isheptaglutamated (αTHF-PG₆) and thus contains a chain of 6 additionalglutamyl groups attached to the glutamyl group of tetrahydrofolate. Insome embodiments, each of the 6 additional glutamyl groups have an alphalinkage. In some embodiments, each of the 6 additional glutamyl groupsin the chain other than the C-terminal glutamyl group or groups have analpha linkage. In some embodiments, 5 of the 6 additional glutamylgroups in the chain have an alpha linkage. In other embodiments, 1, 2,3, 4, or 5, of the 6 additional glutamyl groups have an alpha linkageand the remaining 5, 4, 3, 2, or 1, glutamyl groups, respectively, havea gamma linkage. In other embodiments, 1, 2, 3, 4, or 5 of the 6additional glutamyl groups have an alpha linkage and the remainingnon-C-terminal glutamyl groups are linked to a glutamyl group of themolecule through a gamma linkage. In some embodiments, at least oneadditional glutamyl group has both an alpha linkage and a gamma linkage.In some embodiments, at least one of the 7 glutamyl groups has both analpha linkage and a gamma linkage. In some embodiments, each of the 7glutamyl groups other than the C-terminal glutamyl group or groups havean alpha linkage. In some embodiments, 6 of the 7 glutamyl groups havean alpha linkage. In some embodiments, the alpha heptaglutamated THFcomprises two or more glutamyl groups in the L-form. In furtherembodiments, each of the glutamyl groups of the alpha heptaglutamatedtetrahydrofolate is in the L-form. In other embodiments, the alphaheptaglutamated THF comprises a glutamyl group in the D-form. In furtherembodiments, each of the glutamyl groups of the alpha heptaglutamatedtetrahydrofolate other than the glutamyl group of tetrahydrofolate, isin the D-form. In additional embodiments, the heptaglutamated THFcomprises a glutamyl group in the D-form and two or more glutamyl groupsin the L-form. In some embodiments, the polyglutamate chain is linear.In other embodiments, the polyglutamate chain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate isoctaglutamated (αTHF-PG₇) and thus contains a chain of 7 additionalglutamyl groups attached to the glutamyl group of tetrahydrofolate. Insome embodiments, each of the 7 additional glutamyl groups in the chainother than the C-terminal glutamyl group or groups have an alphalinkage. In some embodiments, 6 of the 7 additional glutamyl groups inthe chain have an alpha linkage. In some embodiments, each of the 7additional glutamyl groups have an alpha linkage. In other embodiments,1, 2, 3, 4, 5, or 6, of the 7 additional glutamyl groups have an alphalinkage and the remaining 6, 5, 4, 3, 2, or 1, glutamyl groups,respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5,or 6 of the 7 additional glutamyl groups have an alpha linkage and theremaining non-C-terminal glutamyl groups are linked to a glutamyl groupof the molecule through a gamma linkage. In some embodiments, at leastone additional glutamyl group has both an alpha linkage and a gammalinkage. In some embodiments, at least one of the 8 glutamyl groups hasboth an alpha linkage and a gamma linkage. In some embodiments, each ofthe 8 glutamyl groups other than the C-terminal glutamyl group or groupshave an alpha linkage. In some embodiments, 7 of the 8 glutamyl groupshave an alpha linkage. In some embodiments, the alpha octaglutamated THFcomprises two or more glutamyl groups in the L-form. In furtherembodiments, each of the glutamyl groups of the alpha octaglutamatedtetrahydrofolate is in the L-form. In other embodiments, the alphaoctaglutamated THF comprises a glutamyl group in the D-form. In furtherembodiments, each of the glutamyl groups of the alpha octaglutamatedtetrahydrofolate other than the glutamyl group of tetrahydrofolate, isin the D-form. In additional embodiments, the octaglutamated THFcomprises a glutamyl group in the D-form and two or more glutamyl groupsin the L-form. In some embodiments, the polyglutamate chain is linear.In other embodiments, the polyglutamate chain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate isnonaglutamated (αTHF-PG₈) and contains a chain of 8 additional glutamylgroups attached to the glutamyl group of tetrahydrofolate. In someembodiments, each of the 8 additional glutamyl groups in the chain otherthan the C-terminal glutamyl group or groups have an alpha linkage. Insome embodiments, 7 of the 8 additional glutamyl groups in the chainhave an alpha linkage. In some embodiments, each of the 8 additionalglutamyl groups have an alpha linkage. In other embodiments, 1, 2, 3, 4,5, 6, or 7, of the 8 additional glutamyl groups have an alpha linkageand the remaining 7, 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively,have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, or 7 ofthe 8 additional glutamyl groups have an alpha linkage and the remainingnon-C-terminal glutamyl groups are linked to a glutamyl group of themolecule through a gamma linkage. In some embodiments, at least oneadditional glutamyl group has both an alpha linkage and a gamma linkage.In some embodiments, at least one of the 9 glutamyl groups has both analpha linkage and a gamma linkage. In some embodiments, each of the 9glutamyl groups other than the C-terminal glutamyl group or groups havean alpha linkage. In some embodiments, 8 of the 9 glutamyl groups havean alpha linkage. In some embodiments, the alpha nonaglutamated THFcomprises two or more glutamyl groups in the L-form. In furtherembodiments, each of the glutamyl groups of the alpha nonaglutamatedtetrahydrofolate is in the L-form. In other embodiments, the alphanonaglutamated THF comprises a glutamyl group in the D-form. In furtherembodiments, each of the glutamyl groups of the alpha nonaglutamatedtetrahydrofolate other than the glutamyl group of tetrahydrofolate, isin the D-form. In additional embodiments, the nonaglutamated THFcomprises a glutamyl group in the D-form and two or more glutamyl groupsin the L-form. In some embodiments, the polyglutamate chain is linear.In other embodiments, the polyglutamate chain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate isdecaglutamated (αTHF-PG₉) (i.e., contains a chain of 9 additionalglutamyl groups attached to the glutamyl group of tetrahydrofolate). Insome embodiments, each of the 9 additional glutamyl groups have an alphalinkage. In some embodiments, each of the 9 additional glutamyl groupsin the chain other than the C-terminal glutamyl group or groups have analpha linkage. In some embodiments, 8 of the 9 additional glutamylgroups in the chain have an alpha linkage. In other embodiments, 1, 2,3, 4, 5, 6, 7, or 8, of the 9 additional glutamyl groups have an alphalinkage and the remaining 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups,respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5,6, 7, or 8 of the 9 additional glutamyl groups have an alpha linkage andthe remaining non-C-terminal glutamyl groups are linked to a glutamylgroup of the molecule through a gamma linkage. In some embodiments, atleast one additional glutamyl group has both an alpha linkage and agamma linkage. In some embodiments, at least one of the 10 glutamylgroups has both an alpha linkage and a gamma linkage. In someembodiments, each of the 10 glutamyl groups other than the C-terminalglutamyl group or groups have an alpha linkage. In some embodiments, 9of the 10 glutamyl groups have an alpha linkage. In some embodiments,the alpha decaglutamated THF comprises two or more glutamyl groups inthe L-form. In further embodiments, each of the glutamyl groups of thealpha decaglutamated tetrahydrofolate is in the L-form. In otherembodiments, the alpha decaglutamated THF comprises a glutamyl group inthe D-form. In further embodiments, each of the glutamyl groups of thealpha decaglutamated tetrahydrofolate other than the glutamyl group oftetrahydrofolate, is in the D-form. In additional embodiments, thedecaglutamated THF comprises a glutamyl group in the D-form and two ormore glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate isundecaglutamated (αTHF-PG₁₀). In some embodiments, each of the 10additional glutamyl groups have an alpha linkage. In some embodiments,each of the 10 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 9 of the 10 additional glutamyl groups in the chain have analpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9, ofthe 10 additional glutamyl groups have an alpha linkage and theremaining 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively,have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9of the 10 additional glutamyl groups have an alpha linkage and theremaining non-C-terminal glutamyl groups are linked to a glutamyl groupof the molecule through a gamma linkage. In some embodiments, at leastone additional glutamyl group has both an alpha linkage and a gammalinkage. In some embodiments, at least one of the 11 glutamyl groups hasboth an alpha linkage and a gamma linkage. In some embodiments, each ofthe 11 glutamyl groups other than the C-terminal glutamyl group orgroups have an alpha linkage. In some embodiments, 10 of the 11 glutamylgroups have an alpha linkage. In some embodiments, the alphaundecaglutamated THF comprises two or more glutamyl groups in theL-form. In further embodiments, each of the glutamyl groups of the alphaundecaglutamated tetrahydrofolate is in the L-form. In otherembodiments, the alpha undecaglutamated THF comprises a D glutamylgroup. In further embodiments, each of the glutamyl groups of the alphaundecaglutamated tetrahydrofolate other than the glutamyl group oftetrahydrofolate, is in the D-form. In additional embodiments, theundecaglutamated THF comprises a glutamyl group in the D-form and two ormore glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate isdodecaglutamated (αTHF-PG₁₁). In some embodiments, each of the 11additional glutamyl groups have an alpha linkage. In some embodiments,each of the 11 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 10 of the 11 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or10, of the 11, additional glutamyl groups have an alpha linkage and theremaining 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups,respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 of the 11 additional glutamyl groups have an alphalinkage and the remaining non-C-terminal glutamyl groups are linked to aglutamyl group of the molecule through a gamma linkage. In someembodiments, at least one additional glutamyl group has both an alphalinkage and a gamma linkage. In some embodiments, at least one of the 12glutamyl groups has both an alpha linkage and a gamma linkage. In someembodiments, each of the 12 glutamyl groups other than the C-terminalglutamyl group or groups have an alpha linkage. In some embodiments, 11of the 12 glutamyl groups have an alpha linkage. In some embodiments,the alpha dodecaglutamated THF comprises two or more glutamyl groups inthe L-form. In further embodiments, each of the glutamyl groups of thealpha dodecaglutamated tetrahydrofolate is in the L-form. In otherembodiments, the alpha dodecaglutamated THF comprises a glutamyl groupin the D-form. In further embodiments, each of the glutamyl groups ofthe alpha dodecaglutamated tetrahydrofolate other than the glutamylgroup of tetrahydrofolate, is in the D-form. In additional embodiments,the dodecaglutamated THF comprises a glutamyl group in the D-form andtwo or more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate istriskaidecaglutamated (αTHF-PG₁₂). In some embodiments, each of the 12additional glutamyl groups have an alpha linkage. In some embodiments,each of the 12 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 11 of the 12 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or 11, of the 12 additional glutamyl groups have an alpha linkage andthe remaining 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups,respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or 11 of the 12 additional glutamyl groups have an alphalinkage and the remaining non-C-terminal glutamyl groups are linked to aglutamyl group of the molecule through a gamma linkage. In someembodiments, at least one additional glutamyl group has both an alphalinkage and a gamma linkage. In some embodiments, at least one of the 13glutamyl groups has both an alpha linkage and a gamma linkage. In someembodiments, each of the 13 glutamyl groups other than the C-terminalglutamyl group or groups have an alpha linkage. In some embodiments, 12of the 13 glutamyl groups have an alpha linkage. In some embodiments,the alpha triskaidecaglutamated THF comprises two or more glutamylgroups in the L-form. In further embodiments, each of the glutamylgroups of the alpha triskaidecaglutamated tetrahydrofolate is in theL-form. In other embodiments, the alpha triskaidecaglutamated THFcomprises a glutamyl group in the D-form. In further embodiments, eachof the glutamyl groups of the alpha triskaidecaglutamatedtetrahydrofolate other than the glutamyl group of tetrahydrofolate, isin the D-form. In additional embodiments, the triskaidecaglutamated THFcomprises a glutamyl group in the D-form and two or more glutamyl groupsin the L-form. In some embodiments, the polyglutamate chain is linear.In other embodiments, the polyglutamate chain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate istetradecaglutamated (αTHF-PG₁₃). In some embodiments, each of the 13additional glutamyl groups have an alpha linkage. In some embodiments,each of the 13 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 12 of the 13 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, or 12, of the 13 additional glutamyl groups have an alpha linkageand the remaining 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamylgroups, respectively, have a gamma linkage. In other embodiments, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the 13 additional glutamyl groupshave an alpha linkage and the remaining non-C-terminal glutamyl groupsare linked to a glutamyl group of the molecule through a gamma linkage.In some embodiments, at least one additional glutamyl group has both analpha linkage and a gamma linkage. In some embodiments, at least one ofthe 14 glutamyl groups has both an alpha linkage and a gamma linkage. Insome embodiments, each of the 14 glutamyl groups other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 13 of the 14 glutamyl groups have an alpha linkage. In someembodiments, the alpha tetradecaglutamated THF comprises two or moreglutamyl groups in the L-form. In further embodiments, each of theglutamyl groups of the alpha tetradecaglutamated tetrahydrofolate is inthe L-form. In other embodiments, the alpha tetradecaglutamated THFcomprises a glutamyl group in the D-form. In further embodiments, eachof the glutamyl groups of the alpha tetradecaglutamated tetrahydrofolateother than the glutamyl group of tetrahydrofolate, is in the D-form. Inadditional embodiments, the tetradecaglutamated THF comprises a glutamylgroup in the D-form and two or more glutamyl groups in the L-form. Insome embodiments, the polyglutamate chain is linear. In otherembodiments, the polyglutamate chain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate ispentadecaglutamated (αTHF-PG₁₄). In some embodiments, each of the 14additional glutamyl groups have an alpha linkage. In some embodiments,each of the 14 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 13 of the 14 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, or 13, of the 14 additional glutamyl groups have an alphalinkage and the remaining 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1,glutamyl groups, respectively, have a gamma linkage. In otherembodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the 14additional glutamyl groups have an alpha linkage and the remainingnon-C-terminal glutamyl groups are linked to a glutamyl group of themolecule through a gamma linkage. In some embodiments, at least oneadditional glutamyl group has both an alpha linkage and a gamma linkage.In some embodiments, at least one of the 15 glutamyl groups has both analpha linkage and a gamma linkage. In some embodiments, each of the 15glutamyl groups other than the C-terminal glutamyl group or groups havean alpha linkage. In some embodiments, 14 of the 15 glutamyl groups havean alpha linkage. In some embodiments, the alpha pentadecaglutamated THFcomprises two or more glutamyl groups in the L-form. In furtherembodiments, each of the glutamyl groups of the alphapentadecaglutamated tetrahydrofolate is in the L-form. In otherembodiments, the alpha pentadecaglutamated THF comprises a glutamylgroup in the D-form. In further embodiments, each of the glutamyl groupsof the alpha pentadecaglutamated tetrahydrofolate other than theglutamyl group of tetrahydrofolate, is in the D-form. In additionalembodiments, the pentadecaglutamated THF comprises a glutamyl group inthe D-form and two or more glutamyl groups in the L-form. In someembodiments, the polyglutamate chain is linear. In other embodiments,the polyglutamate chain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate ishexadecaglutamated (αTHF-PG₁₅). In some embodiments, each of the 15additional glutamyl groups have an alpha linkage. In some embodiments,each of the 15 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 14 of the 15 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or 14, of the 15 additional glutamyl groups have an alphalinkage and the remaining 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or1, glutamyl groups, respectively, have a gamma linkage. In otherembodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 15additional glutamyl groups have an alpha linkage and the remainingnon-C-terminal glutamyl groups are linked to a glutamyl group of themolecule through a gamma linkage. In some embodiments, at least oneadditional glutamyl group has both an alpha linkage and a gamma linkage.In some embodiments, at least one of the 16 glutamyl groups has both analpha linkage and a gamma linkage. In some embodiments, each of the 16glutamyl groups other than the C-terminal glutamyl group or groups havean alpha linkage. In some embodiments, 15 of the 16 glutamyl groups havean alpha linkage. In some embodiments, the alpha hexadecaglutamated THFcomprises two or more glutamyl groups in the L-form. In furtherembodiments, each of the glutamyl groups of the alpha hexadecaglutamatedtetrahydrofolate is in the L-form. In other embodiments, the alphahexadecaglutamated THF comprises a glutamyl group in the D-form. Infurther embodiments, each of the glutamyl groups of the alphahexadecaglutamated tetrahydrofolate other than the glutamyl group oftetrahydrofolate, is in the D-form. In additional embodiments, thehexadecaglutamated THF comprises a glutamyl group in the D-form and twoor more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In other embodiments, the alpha polyglutamated tetrahydrofolate isheptadecaglutamated (αTHF-PG₁₆). In some embodiments, each of the 16additional glutamyl groups have an alpha linkage. In some embodiments,each of the 16 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 15 of the 16 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15, of the 16, additional glutamyl groups have analpha linkage and the remaining 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1, glutamyl groups, respectively, have a gamma linkage. Inother embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15of the 16 additional glutamyl groups have an alpha linkage and theremaining non-C-terminal glutamyl groups are linked to a glutamyl groupof the molecule through a gamma linkage. In some embodiments, at leastone additional glutamyl group has both an alpha linkage and a gammalinkage. In some embodiments, at least one of the 17 glutamyl groups hasboth an alpha linkage and a gamma linkage. In some embodiments, each ofthe 17 glutamyl groups other than the C-terminal glutamyl group orgroups have an alpha linkage. In some embodiments, 16 of the 17 glutamylgroups have an alpha linkage. In some embodiments, the alphaheptadecaglutamated THF comprises two or more glutamyl groups in theL-form. In further embodiments, each of the glutamyl groups of the alphaheptadecaglutamated tetrahydrofolate is in the L-form. In otherembodiments, the alpha heptadecaglutamated THF comprises a D glutamylgroup. In further embodiments, each of the glutamyl groups of the alphaheptadecaglutamated tetrahydrofolate other than the glutamyl group oftetrahydrofolate, is in the D-form. In additional embodiments, theheptadecaglutamated THF comprises a glutamyl group in the D-form and twoor more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate isoctadecaglutamated (αTHF-PG₁₇). In some embodiments, each of the 17additional glutamyl groups have an alpha linkage. In some embodiments,each of the 17 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 16 of the 17 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, or 16, of the 17 additional glutamyl groups have analpha linkage and the remaining 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1, glutamyl groups, respectively, have a gamma linkage.In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,or 16 of the 17 additional glutamyl groups have an alpha linkage and theremaining non-C-terminal glutamyl groups are linked to a glutamyl groupof the molecule through a gamma linkage. In some embodiments, at leastone additional glutamyl group has both an alpha linkage and a gammalinkage. In some embodiments, at least one of the 18 glutamyl groups hasboth an alpha linkage and a gamma linkage. In some embodiments, each ofthe 18 glutamyl groups other than the C-terminal glutamyl group orgroups have an alpha linkage. In some embodiments, 17 of the 18 glutamylgroups have an alpha linkage. In some embodiments, the alphaoctadecaglutamated THF comprises two or more glutamyl groups in theL-form. In further embodiments, each of the glutamyl groups of the alphaoctadecaglutamated tetrahydrofolate is in the L-form. In otherembodiments, the alpha octadecaglutamated THF comprises a glutamyl groupin the D-form. In further embodiments, each of the glutamyl groups ofthe alpha octadecaglutamated tetrahydrofolate other than the glutamylgroup of tetrahydrofolate, is in the D-form. In additional embodiments,the octadecaglutamated THF comprises a glutamyl group in the D-form andtwo or more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate isenneadecaglutamated (αTHF-PG₁₈). In some embodiments, each of the 18additional glutamyl groups have an alpha linkage. In some embodiments,each of the 18 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 17 of the 18 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, or 17, of the 18 additional glutamyl groups havean alpha linkage and the remaining 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,7, 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively, have a gammalinkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, or 17 of the 18 additional glutamyl groups have an alphalinkage and the remaining non-C-terminal glutamyl groups are linked to aglutamyl group of the molecule through a gamma linkage. In someembodiments, at least one additional glutamyl group has both an alphalinkage and a gamma linkage. In some embodiments, at least one of the 19glutamyl groups has both an alpha linkage and a gamma linkage. In someembodiments, each of the 19 glutamyl groups other than the C-terminalglutamyl group or groups have an alpha linkage. In some embodiments, 18of the 19 glutamyl groups have an alpha linkage. In some embodiments,the alpha enneadecaglutamated THF comprises two or more glutamyl groupsin the L-form. In further embodiments, each of the glutamyl groups ofthe alpha enneadecaglutamated tetrahydrofolate is in the L-form. Inother embodiments, the alpha enneadecaglutamated THF comprises a Dglutamyl group. In further embodiments, each of the glutamyl groups ofthe alpha enneadecaglutamated tetrahydrofolate other than the glutamylgroup of tetrahydrofolate, is in the D-form. In additional embodiments,the enneadecaglutamated THF comprises a glutamyl group in the D-form andtwo or more glutamyl groups in the L-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate isicosiglutamated (αTHF-PG₁₉). In some embodiments, each of the 19additional glutamyl groups have an alpha linkage. In some embodiments,each of the 19 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 18 of the 19 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, or 18, of the 19 additional glutamyl groupshave an alpha linkage and the remaining 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively, have agamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, or 18 of the 19 additional glutamyl groups havean alpha linkage and the remaining non-C-terminal glutamyl groups arelinked to a glutamyl group of the molecule through a gamma linkage. Insome embodiments, at least one additional glutamyl group has both analpha linkage and a gamma linkage. In some embodiments, at least one ofthe 20 glutamyl groups has both an alpha linkage and a gamma linkage. Insome embodiments, each of the 20 glutamyl groups other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 19 of the 20 glutamyl groups have an alpha linkage. In someembodiments, the alpha icosiglutamated THF comprises two or moreglutamyl groups in the L-form. In further embodiments, each of theglutamyl groups of the alpha icosiglutamated tetrahydrofolate is in theL-form. In other embodiments, the alpha icosiglutamated THF comprises aglutamyl group in the D-form. In further embodiments, each of theglutamyl groups of the alpha icosiglutamated tetrahydrofolate other thanthe glutamyl group of tetrahydrofolate, is in the D-form. In additionalembodiments, the icosiglutamated THF comprises a glutamyl group in theD-form and two or more glutamyl groups in the L-form. In someembodiments, the polyglutamate chain is linear. In other embodiments,the polyglutamate chain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate isicosikaihenaglutamated (αTHF-PG₂₀). In some embodiments, each of the 20additional glutamyl groups have an alpha linkage. In some embodiments,each of the 20 additional glutamyl groups in the chain other than theC-terminal glutamyl group or groups have an alpha linkage. In someembodiments, 19 of the 20 additional glutamyl groups in the chain havean alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, or 19, of the 20 additional glutamylgroups have an alpha linkage and the remaining 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups,respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the 20additional glutamyl groups have an alpha linkage and the remainingnon-C-terminal glutamyl groups are linked to a glutamyl group of themolecule through a gamma linkage. In some embodiments, at least oneadditional glutamyl group has both an alpha linkage and a gamma linkage.In some embodiments, at least one of the 21 glutamyl groups has both analpha linkage and a gamma linkage. In some embodiments, each of the 21glutamyl groups other than the C-terminal glutamyl group or groups havean alpha linkage. In some embodiments, 20 of the 21 glutamyl groups havean alpha linkage. In some embodiments, the alpha icosikaihenaglutamatedTHF comprises two or more glutamyl groups in the L-form. In furtherembodiments, each of the glutamyl groups of the alphaicosikaihenaglutamated tetrahydrofolate is in the L-form. In otherembodiments, the alpha icosikaihenaglutamated THF comprises a glutamylgroup in the D-form. In further embodiments, each of the glutamyl groupsof the alpha icosikaihenaglutamated tetrahydrofolate other than theglutamyl group of tetrahydrofolate, is in the D-form. In additionalembodiments, the icosikaihenaglutamated THF comprises a glutamyl groupin the D-form and two or more glutamyl groups in the L-form. In someembodiments, the polyglutamate chain is linear. In other embodiments,the polyglutamate chain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate containsa chain of 4-7 glutamyl groups attached to tetrahydrofolate (i.e.,αTHF-PGn, wherein n=4-7) and each of the 4-7 attached glutamyl groupshave an alpha linkage. In some embodiments, the alpha polyglutamatedtetrahydrofolate contains a chain of 4-7 glutamyl groups attached totetrahydrofolate (i.e., αTHF-PGn, wherein n=4-7) and each of the 4-7attached glutamyl groups other than the C-terminal glutamyl group orgroups has an alpha linkage. In some embodiments, each of the 4-7attached glutamyl groups is in the L-form. In other embodiments, each ofthe 4-7 attached glutamyl groups is in the D-form. In other embodiments,the 4-7 attached glutamyl groups are in the L-form and the D-form. Insome embodiments, the polyglutamate chain is linear. In otherembodiments, the polyglutamate chain is branched.

In one embodiment, the alpha polyglutamated tetrahydrofolate istetraglutamated and each of the 3 glutamyl groups in the polyglutamatechain attached to the tetrahydrofolate contains an alpha linkage. In oneembodiment, the alpha polyglutamated tetrahydrofolate is tetraglutamatedand each of the 3 glutamyl groups in the polyglutamate chain attached tothe tetrahydrofolate other than the C-terminal glutamyl group or groupscontains an alpha linkage. In some embodiments, each of the 4 glutamylgroups is in the L-form. In some embodiments, each of the glutamylgroups in the alpha tetraglutamated tetrahydrofolate other than theglutamyl group of tetrahydrofolate, is in the D-form. In otherembodiments, at least two glutamyl groups in the alpha tetraglutamatetetrahydrofolate are in the L-form and at least one glutamyl group is inthe D-form. In some embodiments, the polyglutamate chain is linear. Inother embodiments, the polyglutamate chain is branched.

In one embodiment, the alpha polyglutamated tetrahydrofolate ispentaglutamated and each of the 4 glutamyl groups in the polyglutamatechain attached to the tetrahydrofolate contains an alpha linkage. In oneembodiment, the alpha polyglutamated tetrahydrofolate is pentaglutamatedand each of the 4 glutamyl groups in the polyglutamate chain attached tothe tetrahydrofolate other than the C-terminal glutamyl group or groupscontains an alpha linkage. In some embodiments, each of the 4 glutamylgroups is in the L-form. In some embodiments, each of the glutamylgroups in the alpha pentaglutamated tetrahydrofolate other than theglutamyl group of tetrahydrofolate, is in the D-form. In otherembodiments, at least two glutamyl groups in the alpha pentaglutamatedtetrahydrofolate are in the L-form and at least one glutamyl group is inthe D-form. In some embodiments, the polyglutamate chain is linear. Inother embodiments, the polyglutamate chain is branched.

In one embodiment, the alpha polyglutamated tetrahydrofolate ishexaglutamated and each of the 5 glutamyl groups in the polyglutamatechain attached to the tetrahydrofolate contains an alpha linkage. In oneembodiment, the alpha polyglutamated tetrahydrofolate is hexaglutamatedand each of the 5 glutamyl groups in the polyglutamate chain attached tothe tetrahydrofolate other than the C-terminal glutamyl group or groupscontains an alpha linkage. In some embodiments, each of the 5 glutamylgroups is in the L-form. In some embodiments, each of the glutamylgroups in the alpha hexaglutamated tetrahydrofolate other than theglutamyl group of tetrahydrofolate, is in the D-form. In otherembodiments, at least two glutamyl groups in the alpha hexaglutamatedtetrahydrofolate are in the L-form and at least one glutamyl group is inthe D-form. In some embodiments, the polyglutamate chain is linear. Inother embodiments, the polyglutamate chain is branched.

In another embodiment, the alpha polyglutamated tetrahydrofolate isheptaglutamated and each of the 6 glutamyl groups in the polyglutamatechain attached to the tetrahydrofolate contains an alpha linkage. Inanother embodiment, the alpha polyglutamated tetrahydrofolate isheptaglutamated and each of the 6 glutamyl groups in the polyglutamatechain attached to the tetrahydrofolate other than the C-terminalglutamyl group or groups contains an alpha linkage. In some embodiments,each of the 6 glutamyl groups is in the L-form. In some embodiments,each of the glutamyl groups in the alpha heptaglutamatedtetrahydrofolate other than the glutamyl group of tetrahydrofolate, isin the D-form. In other embodiments, at least two glutamyl groups in thealpha heptaglutamated tetrahydrofolate are in the L-form and at leastone glutamyl group is in the D-form. In some embodiments, thepolyglutamate chain is linear. In other embodiments, the polyglutamatechain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate (αPTHF)contains a total of 1-15, 1-10, 2-15, 2-10, 3-15, 3-10, 3-6, 3-5, 4-10,4-7, or 4-6, glutamyl groups including the glutamyl group intetrahydrofolate, or any range therein between. In some embodiments,each of the glutamyl groups in the αPTHF other than the glutamyl groupof tetrahydrofolate have an alpha linkage. In some embodiments, each ofthe glutamyl groups in the αPTHF other than the C-terminal glutamylgroup or groups and the glutamyl group of tetrahydrofolate has an alphalinkage. In some embodiments, each of the glutamyl groups in the αPTHFother than the C-terminal glutamyl group or groups has an alpha linkage.In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14,of the glutamyl groups in the αPTHF have an alpha linkage. In someembodiments, the αPTHF comprises glutamyl groups in the L-form and theD-form. In further embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, or 14, of the glutamyl groups in the αPTHF have an alpha linkage and13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or none, of the glutamylgroups, respectively, has a gamma linkage. In some embodiments, each ofthe glutamyl groups in the polyglutamate structure of the polyglutamatedtetrahydrofolate is in the L-form. In some embodiments, each of theglutamyl groups in the αPTHF other than the glutamyl group oftetrahydrofolate is in the D-form. In one embodiment, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15, of the glutamyl groups in the αPTHFis in the L-form. In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or 14, of the glutamyl groups in the αPTHF is in the D-form.In some embodiments, the polyglutamate chain is linear. In otherembodiments, the polyglutamate chain is branched.

In some embodiments, the alpha polyglutamated tetrahydrofolate (αPTHF)contains a total of 2-20, 2-15, 2-10, 2-5, glutamyl groups including theglutamyl group in tetrahydrofolate, or any range therein between. Insome embodiments, each of the glutamyl groups in the αPTHF other thanthe glutamyl group of tetrahydrofolate, have an alpha linkage. In someembodiments, each of the glutamyl groups in the αPTHF other than theC-terminal glutamyl group or groups and the glutamyl group oftetrahydrofolate has an alpha linkage. In some embodiments, each of theglutamyl groups in the αPTHF other than the C-terminal glutamyl group orgroups has an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, of the glutamyl groupshave an alpha linkage. In some embodiments, the αPTHF contains two ormore glutamyl groups having a gamma linkage. In further embodiments, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, ofthe glutamyl groups in the αPTHF other than the glutamyl group oftetrahydrofolate have an alpha linkage and 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or none, of the glutamyl groups,respectively, has a gamma linkage. In some embodiments, each of theglutamyl groups in the αPTHF is in the L-form. In some embodiments, eachof the glutamyl groups in the αPTHF other than the glutamyl group oftetrahydrofolate is in the D-form. In one embodiment, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, of the glutamylgroups in the αPTHF are in the L-form. In another embodiment, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, glutamylgroups in the αPTHF is in the D-form.

In some embodiments, the alpha polyglutamated tetrahydrofolate containsa total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15,glutamyl groups in addition to the glutamyl group in tetrahydrofolate).In further embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,or 15, of the additional glutamyl groups have an alpha linkage. Inadditional embodiments, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or1, of the glutamyl groups in the alpha polyglutamated tetrahydrofolatehave a gamma linkage. In some embodiments, at least one glutamyl grouphas both an alpha linkage and a gamma linkage. In some embodiments, theglutamyl group in tetrahydrofolate has an alpha linkage. In someembodiments, the glutamyl group in tetrahydrofolate has both an alphalinkage and a gamma linkage.

In some embodiments, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, or 15, glutamyl groups in the polyglutamated alphatetrahydrofolate are in the L-form, the D-form, or in the L-form and theD-form. In some embodiments, each of the glutamyl groups of thepolyglutamated alpha tetrahydrofolate is in the L-form. In otherembodiments, each of the glutamyl groups of the polyglutamated alphatetrahydrofolate other than the glutamyl group of tetrahydrofolate is inthe D-form. In alternative embodiments, at least two of the glutamylgroups in the polyglutamated alpha tetrahydrofolate are in the L-formand at least one of the glutamyl groups in the polyglutamated alphatetrahydrofolate is in the D-form. In some embodiments, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, glutamyl groups in thepolyglutamated alpha tetrahydrofolate are in the L-form. In otherembodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, glutamylgroups in the polyglutamated alpha tetrahydrofolate are in the D-form.In some embodiments, at least one glutamyl group has both an alphalinkage and a gamma linkage.

In additional embodiments, the polyglutamated alpha tetrahydrofolatecontains 20-100, 20-75, 20-50, 20-40, 20-30, 20-25, or more than 100,alpha glutamyl groups, or any range therein between. In someembodiments, each of the glutamyl groups of the αPTHF is in the L-form.In other embodiments, each of the glutamyl groups of the αPTHF otherthan the glutamyl group of tetrahydrofolate is in the D-form. Inalternative embodiments, at least two of the glutamyl groups in theαPTHF are in the L-form and at least one of the glutamyl groups in theαPTHF is in the D-form. In some embodiments, at least one glutamyl grouphas both an alpha linkage and a gamma linkage.

In additional embodiments, the provided compositions comprise a αPTHFthat contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20, glutamyl groupsthat have alpha linkages. In some embodiments, the αPTHF contains 1, 2,3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20, glutamyl groups in the L-form. Insome embodiments, the αPTHF contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10,or 1-20, glutamyl groups in the D-form. In some embodiments, the αPTHFcontains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20, glutamyl groups inthe L-form and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 or 1-20, glutamylgroups in the D-form. In other embodiments, the polyglutamated alphatetrahydrofolate contains at least 1 glutamyl group that has both analpha linkage and a gamma linkage. In some embodiments, thepolyglutamated alpha tetrahydrofolate contains 1, 2, 3, 4, 5, 6, 7, 8,9, 1-10, or more than 10 glutamyl groups that have both an alpha linkageand a gamma linkage.

In some embodiments, the alpha-polyglutamated tetrahydrofolate containsa least 1 glutamyl group having an alpha linkage and contains 2, 3, 4,5, 6, 7, 8, 9, 1-10, 1-20, or more, glutamyl groups having a gammalinkage. For example, in some embodiments, the polyglutamated alphatetrahydrofolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10, L-alphaglutamyl group linkages and further contains 1, 2, 3, 4, 5, 6, 7, 8, 9,or 1-10, L-gamma glutamyl group linkages. In some further embodiments,the polyglutamated alpha tetrahydrofolate contains 1, 2, 3, 4, 5, 6, 7,8, 9, or 1-10, L-alpha glutamyl group linkages and further contains 1,2, 3, 4, 5, 6, 7, 8, 9, or 1-10, D-gamma glutamyl group linkages. Inadditional further embodiments, the polyglutamated alphatetrahydrofolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10, D-alphaglutamyl group linkages and further contains 1, 2, 3, 4, 5, 6, 7, 8, 9,or 1-10, D-gamma glutamyl group linkages. In other further embodiments,the polyglutamated alpha tetrahydrofolate contains 1, 2, 3, 4, 5, 6, 7,8, 9, or 1-10, D-gamma glutamyl group linkages and further contains 1,2, 3, 4, 5, 6, or 1-10, L-gamma glutamyl group linkages. In otherembodiments, the polyglutamated alpha tetrahydrofolate contains at least1 glutamyl group that has both an alpha linkage and a gamma linkage. Insome embodiments, the polyglutamated alpha tetrahydrofolate contains 1,2, 3, 4, 5, 6, 7, 8, 9, 1-10, or more than 10, glutamyl groups that haveboth an alpha linkage and a gamma linkage.

In some embodiments, the αPTHF composition provided herein is capable ofadding one or more additional glutamyl groups that, is the compositionis able to act as a substrate for by FPGS (folylpolyglutamatesynthetase). Reagents and assays and reagents for determining theability of a αPTHF composition to act as a substrate for FPGS (e.g.,human FPGS, or rat liver FPGS) are readily available and can routinelybe performed.

In some embodiments, the rate of uptake of naked alpha PTHF compositionsdisclosed herein (e.g., alpha PTHF that is not associated with adelivery vehicle) are taken up by hepatic cells at a significantlyreduced rate compared to the uptake rate of tetrahydrofolate under thesame physiological conditions. In some embodiments, the rate of hepaticcell uptake of the naked alpha PTHF composition is less than 30%, 20%,15%, or 10% compared to the rate of tetrahydrofolate. In furtherembodiments, the rate of the efflux (transport out) of alpha PTHFcompositions disclosed herein from hepatic-cells occurs at a rate thatis significantly reduced compared to tetrahydrofolate (e.g., less than30%, 20%, 15%, or 10%) compared to the rate of tetrahydrofolate.

In some embodiments, an alpha polyglutamated tetrahydrofolatecomposition provided herein is more cytotoxic to hyperproliferativecells than tetrahydrofolate. In some embodiments the hyperproliferativecells are cancer cells. In some embodiments, the hyperproliferativecells a colorectal carcinoma cells, colon cancer cells, breast cancercells, or ovarian cancer cells. In some embodiments, the cancer cellsare mesothelioma cells or non-small cell lung carcinoma cells. In someembodiments, cytotoxicity is measured in an in vitro assay. In someembodiments, the alpha polyglutamated tetrahydrofolate is ahexaglutamated tetrahydrofolate.

In some embodiments, an alpha polyglutamated tetrahydrofolatecomposition provided herein has lower toxic side effects thantetrahydrofolate. In some embodiments, the alpha polyglutamatedtetrahydrofolate composition provided herein is less toxic tonon-hyperproliferative cells than tetrahydrofolate. In some embodiments,the alpha polyglutamated tetrahydrofolate composition provided herein isless toxic to neutrophils, liver cells, or to colon epithelium cellsthan tetrahydrofolate. In some embodiments, the neutrophils humanneutrophils, differentiating human neutrophils, or neutrophilsdifferentiated from CD34+ cells. In some embodiments, the liver cellsare AML12 liver cells. In some embodiments, the colon epithelium cellsare CCD841 colon epithelium cells. In some embodiments, the toxicity ismeasured in an in vitro assay. In some embodiments, the alphapolyglutamated tetrahydrofolate is a hexaglutamated tetrahydrofolate.

In some embodiments, an alpha polyglutamated tetrahydrofolatecomposition provided herein has lower toxic side effects than totetrahydrofolate. In some embodiments, an alpha polyglutamatedtetrahydrofolate composition provided herein causes fewer or less severetoxic side effects in an vivo assay than tetrahydrofolate. In someembodiments, the in vivo assay is an in vivo murine model. In someembodiments, an alpha polyglutamated tetrahydrofolate compositionprovided herein causes fewer or less severe hematological or hepatictoxic side effects than tetrahydrofolate. In some embodiments,hematological side effects are assessed by measuring mean neutrophil,mean white blood cell or mean platelet counts. In some embodiments,hepatic toxic side effects are assessed by measuring serum aspartatetransaminase (AST), serum alanine transaminase (ALT), and/or serumalbumin levels. In some embodiments, the in vivo assay comprisesadministering 40 mg/kg or 80 mg/kg of the alpha polyglutamatedtetrahydrofolate composition once weekly for 4 weeks. In someembodiments, the alpha polyglutamated tetrahydrofolate is ahexaglutamated tetrahydrofolate.

In some embodiments, treatment with an alpha polyglutamatedtetrahydrofolate composition provided herein does not induce significanthematological or hepatic toxic side effects in an in vivo murine model.In some embodiments, hematological side effects are assessed bymeasuring mean neutrophil, mean white blood cell or mean plateletcounts. In some embodiments, hepatic toxic side effects are assessed bymeasuring serum aspartate transaminase (AST), serum alanine transaminase(ALT), and/or serum albumin levels. In some embodiments, an alphapolyglutamated tetrahydrofolate composition provided herein does notsignificantly decrease mean neutrophil, mean white blood cell or meanplatelet counts. In some embodiments, an alpha polyglutamatedtetrahydrofolate composition provided herein does not significantlyincrease serum aspartate transaminase (AST) and serum alaninetransaminase (ALT) levels. In some embodiments, an alpha polyglutamatedtetrahydrofolate composition provided herein does not significantlydecrease serum albumin levels. In some embodiments, the in vivo assaycomprises administering 40 mg/kg or 80 mg/kg of the alpha polyglutamatedtetrahydrofolate composition once weekly for 4 weeks. In someembodiments, the alpha polyglutamated tetrahydrofolate is ahexaglutamated tetrahydrofolate.

In some embodiments, the αPTHF compositions do not contain a fluorineatom. In some embodiments, the αPTHF compositions do not contain a4-fluoroglutamyl group.

Polyglutamated alpha tetrahydrofolate (αPTHF) compositions and theiruses are further disclosed in U.S. Appl. Nos. 62/374,458, 62/583,432,62/627,741, 62/630,820 and 62/630,821, 62/630,824, 62/630,825, Ser. Nos.15/675,695, and 15/675,701; and Intl. Appl. Nos. PCT/US2017/046666, andPCT/US2017/046667; the contents of each of which is herein incorporatedby reference in its entirety.

A. Polyglutamated Tetrahydrofolate Analogs and Derivatives

The disclosure also encompasses αPTHF derivatives and analogs. Thecompositions and methods disclosed herein are envisioned to apply to anyand every known derivative or analog of tetrahydrofolate that ispolyglutamated. In some embodiments the polyglutamated tetrahydrofolateanalog or derivative composition prepared and used according to thedisclosed compositions and methods is depicted in FIGS. 1I-1J. In someembodiments the analog corresponds to a modified form oftetrahydrofolate wherein the glutamyl group of tetrahydrofolate is notlinked to the remainder of tetrahydrofolate molecule through a gammapeptide linkage. In some embodiments, the analog is a variant form oftetrahydrofolate wherein the glutamyl group of tetrahydrofolate in inthe D-form. In some embodiments, the polyglutamated form oftetrahydrofolate, or polyglutamated tetrahydrofolate analog orderivative is not fluorinated.

In additional embodiments, the polyglutamated alpha tetrahydrofolatederivative or analog has a variant polyglutamate chain. In someembodiments the polyglutamate chain contains one or more natural orsynthetic residues other than glutamate. In some embodiments thepolyglutamate chain contains one or more glutamyl groups that do notcontain an amide linkage. In other embodiments, one or more of theglutamyl groups of the polyglutamate chain is derivatized.

B. THF-PG Synthesis

The tetrahydrofolate polyglutamate compositions provided herein may beobtained by following synthetic procedures using available reagents andsynthetic intermediates. The addition of glutamyl residues to theglutamyl residues of tetrahydrofolate can be accomplished usingsynthetic procedures known in the art. In some embodiments, glutamylresidues are added serially to the glutamyl residue of tetrahydrofolate.In additional embodiments, polyglutamates are added to the glutamylreside of tetrahydrofolate using “click chemistry” methods or otherbioconjugate chemistries known to those in the art. Alternatively apeptide of glutamyl residues can be generated of the desired length andadded to a precursor of tetrahydrofolate which does not have a glutamylresidue. The peptide can be produced using synthetic procedures known inthe art. In some embodiments, an initial glutamyl residue is bonded towang resin and additional glutamyl residues are added serially via solidphase peptide synthesis using F-moc chemistry. After the final glutamylresidue is added the tetrahydrofolate precursor is coupled to thepeptide and the molecule is cleaved from the resin.

The addition of glutamyl residues to the glutamyl residues oftetrahydrofolate can be accomplished using synthetic procedures known inthe art. In some embodiments, glutamyl residues are added serially tothe glutamyl residue of tetrahydrofolate. In additional embodiments,polyglutamates are added to the glutamyl reside of tetrahydrofolateusing “click chemistry” methods or other bioconjugate chemistries knownto those in the art. Alternatively a peptide of glutamyl residues can begenerated of the desired length and added to a precursor oftetrahydrofolate which does not have a glutamyl residue. The peptide canbe produced using synthetic procedures known in the art. In someembodiments, an initial glutamyl residue is bonded to wang resin andadditional glutamyl residues are added serially via solid phase peptidesynthesis using F-moc chemistry. After the final glutamyl residue isadded the tetrahydrofolate precursor is coupled to the peptide and themolecule is cleaved from the resin.

C. Tetrahydrofolate-PG Complexes

The inventors have surprising found that polyglutamated antifolates thatshare similar structural and chemical features with tetrahydrofolate(αPTHF) are able to form complexes with other compositions includingtherapeutic agents, including cytotoxic compounds such as platinum-basedcompounds. Accordingly, in some embodiments, the disclosure provides acomplex of a αPTHF (e.g., a αPTHF disclosed herein) and a therapeuticagent or a salt or acid thereof.

In some embodiments, the αPTHF/complex comprise αPTHF and a therapeuticagent. In some embodiments, the therapeutic agent is a cytotoxiccompound such as a chemotherapeutic agent. In further embodiments, theαPTHF/complex contains a platinum-based drug such as platinum-basedchemotherapeutic agent (e.g., cisplatin, carboplatin and oxaliplatin).In other embodiments, the αPTHF/complex contains a taxane-basedchemotherapeutic agent (e.g., paclitaxel and docetaxel). In otherembodiments, the αPTHF/complex contains a cyclodextrin. In furtherembodiments, the αPTHF/complex is encapsulated in a liposome

In some embodiments, the disclosure provides a composition comprising acomplex of a αPTHF and a therapeutic agent or a salt or acid thereof. Infurther embodiments, the αPTHF/therapeutic agent complex comprises oneor more αPTHF containing 2-150, 2-100, 2-75, 2-50, 2-24, 2-30, 2-20,2-19, 2-15, 2-10, or 2-5, glutamyl groups. In some embodiments, theαPTHF/therapeutic agent complex comprises one or more αPTHF containing3-10, 3-9, 3-8, or 3-7, glutamyl groups, or any range therein between.In other embodiments, the αPTHF/therapeutic agent complex comprises oneor more αPTHF containing 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5, glutamylgroups, or any range therein between. In one particular embodiment, thecomplex comprises one or more αPTHF containing 3-10 glutamyl groups. Infurther embodiments, the αPTHF/therapeutic agent complex comprises oneor more αPTHF containing 3-7 glutamyl groups. In another embodiment, theαPTHF/therapeutic agent complex comprises one or more αPTHF containing 5glutamyl groups. In another embodiment, the αPTHF/therapeutic agentcomplex comprises one or more αPTHF containing 6 glutamyl groups. Insome embodiments, the therapeutic agent is a cytotoxic compound or asalt or acid thereof. In a further embodiment, the therapeutic agent isa chemotherapeutic agent or a salt or acid thereof. In anotherembodiment, the therapeutic agent is a platinum-based drug. In anotherembodiment, the therapeutic agent is a taxane-based drug. In additionalembodiments, the molar ratio of αPTHF/therapeutic agent in the complexis in the range 1-10:1. In some embodiments, the molar ratio ofαPTHF/therapeutic agent in the complex is: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1,19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratioof αPTHF/therapeutic agent in the complex is in the range 1:1-20,1:1-10, or 1:2-8, or any range therein between. In some embodiments, themolar ratio of αPPTHF/therapeutic agent is: 1:1, 1:2, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In some embodiments, theαPTHF/therapeutic agent complex is encapsulated in a liposome (e.g., asdescribed herein or otherwise known in the art).

In an alternative embodiment, the αPTHF complex comprises αPTHF andcyclodextrin. In some embodiments, the molar ratio of αPTHF (e.g., αPTHFsalt)/cyclodextrin in the complex is in the range 1-20:1, or any rangetherein between. In some embodiments, the molar ratio ofαPTHF/cyclodextrin in the complex is in the range 1-10:1, or any rangetherein between. In further embodiments, the molar ratio ofαPTHF/cyclodextrin in the complex is in the range 2-8:1, or any rangetherein between. In some embodiments, the molar ratio ofαPTHF/cyclodextrin in the complex is: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1,8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1,20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio ofαPTHF/cyclodextrin in the complex is in the range 1:1-20, 1:1-10, or1:2-8, or any range therein between. In some embodiments, the molarratio of αPTHF/cyclodextrin is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:(21-50), or 1:>50. In some embodiments, the αPTHF/cyclodextrin complexis encapsulated in a liposome (e.g., as described herein or otherwiseknown in the art).

In some embodiments, the disclosure provides a composition comprising aαPTHF/platinum-based chemotherapeutic agent complex. In someembodiments, the platinum-based chemotherapeutic agent is selected fromthe group consisting of: cisplatin, carboplatin, and oxaliplatin, or asalt or acid thereof. In other embodiments, the αPTHF/platinum-basedchemotherapeutic agent complex comprises an analog of a cisplatin,carboplatin, oxaliplatin, or a salt or acid thereof. In someembodiments, the molar ratio of αPTHF/platinum-based agent in thecomplex is in the range 1-20:1, or any range therein between. In someembodiments, the molar ratio of αPTHF/platinum-based agent in thecomplex is in the range 1-10:1, or any range therein between. In furtherembodiments, the molar ratio of αPTHF/platinum-based agent in thecomplex is in the range 2-8:1, or any range therein between. In someembodiments, the molar ratio of αPTHF/platinum-based agent in thecomplex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1,or >50:1. In other embodiments, the molar ratio of αPTHF/platinum-basedagent in the complex is in the range 1:1-20, 1:1-10, or 1:2-8, or anyrange therein between. In some embodiments, the molar ratio ofαPTHF/platinum-based agent is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:(21-50), or 1:>50. In additional embodiments, the αPTHF/platinum-basedagent complex is encapsulated in a liposome (e.g., as described hereinor otherwise known in the art).

In additional embodiments, the αPTHF/platinum-based chemotherapeuticagent complex comprises an analog of a cisplatin, carboplatin,oxaliplatin, or a salt or acid thereof. In some embodiments, the molarratio of αPTHF/platinum-based analog in the complex is in the range1-20:1, or any range therein between. In some embodiments, the molarratio of αPTHF/platinum-based analog in the complex is in the range1-10:1, or any range therein between. In further embodiments, the molarratio of αPTHF/platinum-based agent in the complex is in the range2-8:1, or any range therein between. In some embodiments, the molarratio of αPTHF/platinum-based analog in the complex is 1:1, 2:1, 3:1,4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1,17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, themolar ratio of αPTHF/platinum-based analog in the complex is in therange 1:1-20, 1:1-10, or 1:2-8, or any range therein between. In someembodiments, the molar ratio of αPTHF/platinum-based analog is: 1:1,1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14,1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additionalembodiments, the αPTHF/platinum-based analog complex is encapsulated ina liposome (e.g., as described herein or otherwise known in the art).

In further embodiments, the disclosure provides a complex containingαPTHF and cisplatin or a salt or acid thereof. In some embodiments, themolar ratio of αPTHF/cisplatin (or cisplatin salt or acid) in thecomplex is in the range 1-20:1, or any range therein between. In someembodiments, the molar ratio of αPTHF/cisplatin (or cisplatin salt oracid) in the complex is in the range 1-10:1, or any range thereinbetween. In further embodiments, the molar ratio of αPTHF/cisplatin (orcisplatin salt or acid) in the complex is in the range 2-8:1, or anyrange therein between. In some embodiments, the molar ratio ofαPTHF/cisplatin (or cisplatin salt or acid) in the complex is 1:1, 2:1,3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1,16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments,the molar ratio of αPTHF/cisplatin (or cisplatin salt or acid) in thecomplex is in the range 1:1-20, 1:1-10, or 1:2-8, or any range thereinbetween. In some embodiments, the molar ratio of αPTHF/cisplatin (orcisplatin salt or acid) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:(21-50), or 1:>50. In additional embodiments, the αPTHF/cisplatin (orcisplatin salt or acid) complex is encapsulated in a liposome (e.g., asdescribed herein or otherwise known in the art).

In another embodiment, the disclosure provides a complex containingαPTHF and carboplatin or a salt or acid thereof. In some embodiments,the molar ratio of αPTHF/carboplatin (or carboplatin salt or acid) inthe complex is in the range 1-20:1, or any range therein between. Infurther embodiments, the molar ratio of αPTHF/carboplatin (orcarboplatin salt or acid) in the complex is in the range 1-10:1, or anyrange therein between. In further embodiments, the molar ratio ofαPTHF/carboplatin (or carboplatin salt or acid) in the complex is in therange 2-8:1, or any range therein between. In some embodiments, themolar ratio of αPTHF/carboplatin (or carboplatin salt or acid) in thecomplex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1,or >50:1. In other embodiments, the molar ratio of αPTHF/carboplatin (orcarboplatin salt or acid) in the complex is in the range 1:1-20, 1:1-10,or 1:2-8, or any range therein between. In some embodiments, the molarratio of αPTHF/carboplatin (or carboplatin salt or acid) is: 1:1, 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15,1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additionalembodiments, the αPTHF/carboplatin (or carboplatin salt or acid) complexis encapsulated in a liposome (e.g., as described herein or otherwiseknown in the art).

In another embodiment, the disclosure provides a complex containingαPTHF and oxaliplatin, or a salt or acid thereof. In some embodiments,the molar ratio of αPTHF/oxaliplatin (or oxaliplatin salt or acid) inthe complex is in the range 1-20:1, or any range therein between. Infurther embodiments, the molar ratio of αPTHF/oxaliplatin (oroxaliplatin salt or acid) in the complex is in the range 1-10:1, or anyrange therein between. In further embodiments, the molar ratio ofαPTHF/oxaliplatin (or oxaliplatin salt or acid) in the complex is in therange 2-8:1, or any range therein between. In some embodiments, themolar ratio of αPTHF/oxaliplatin (or oxaliplatin salt or acid) in thecomplex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1,or >50:1. In other embodiments, the molar ratio of αPTHF/oxaliplatin (oroxaliplatin salt or acid) in the complex is in the range 1:1-20, 1:1-10,or 1:2-8, or any range therein between. In some embodiments, the molarratio of αPTHF/oxaliplatin (or oxaliplatin salt or acid) is: 1:1, 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15,1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additionalembodiments, the αPTHF/oxaliplatin (or oxaliplatin salt or acid) complexis encapsulated in a liposome (e.g., as described herein or otherwiseknown in the art).

In additional embodiments, the disclosure provides a complex comprisingαPTHF and a platinum-based chemotherapeutic agent (platinum) selectedfrom the group consisting of: nedaplatin, heptaplatin, lobaplatin,stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin,spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin,zeniplatin, platinum-triamine, traplatin, enloplatin, JM216, NK121,CI973, DWA 2114R, NDDP, and dedaplatin, or a salt or acid thereof. Inother embodiments, the αPTHF/platinum-based chemotherapeutic agentcomplex comprises an analog of nedaplatin, heptaplatin, lobaplatin,stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin,spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin,zeniplatin, platinum-triamine, traplatin, enloplatin, JM216, NK121,CI973, DWA 2114R, NDDP, or dedaplatin, or a salt or acid thereof. Insome embodiments, the molar ratio of αPTHF/platinum (or platinum salt oracid) in the complex is in the range 1-20:1, or any range thereinbetween. In further embodiments, the molar ratio of αPTHF/platinum (orplatinum salt or acid) in the complex is in the range 1-10:1, or anyrange therein between. In further embodiments, the molar ratio ofαPTHF/platinum (or platinum salt or acid) in the complex is in the range2-8:1, or any range therein between. In some embodiments, the molarratio of αPTHF/platinum (or platinum salt or acid) in the complex is1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1,14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In otherembodiments, the molar ratio of αPTHF/platinum (or platinum salt oracid) in the complex is in the range 1:1-20, 1:1-10, or 1:2-8, or anyrange therein between. In some embodiments, the molar ratio ofαPTHF/platinum (or platinum salt or acid) is: 1:1, 1:2, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments, theαPTHF/platinum (or salt or acid or analog thereof) complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In some embodiments, the disclosure provides a composition comprising aαPTHF/taxane-based chemotherapeutic agent (taxane) complex. In someembodiments, the taxane-based chemotherapeutic agent is selected fromthe group consisting of: paclitaxel (PTX), docetaxel (DTX), larotaxel(LTX), and cabazitaxel (CTX), or a salt or acid thereof. In someembodiments, the molar ratio of αPTHF/taxane-based agent in the complexis in the range 1-20:1, or any range therein between. In furtherembodiments, the molar ratio of αPTHF/taxane (or taxane salt or acid) inthe complex is in the range 1-10:1, or any range therein between. Infurther embodiments, the molar ratio of αPTHF/taxane (or taxane salt oracid) in the complex is in the range 2-8:1, or any range thereinbetween. In some embodiments, the molar ratio of αPTHF/taxane (or taxanesalt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In other embodiments, the molar ratio ofαPTHF/taxane (or taxane salt or acid) in the complex is in the range1:1-20, 1:1-10, or 1:2-8, or any range therein between. In someembodiments, the molar ratio of a αPTHF/taxane (or taxane salt or acid)is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13,1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. Inadditional embodiments, the αPTHF/taxane-based agent complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In additional embodiments, the disclosure provides a complex comprisingαPTHF and paclitaxel (PTX), or a salt or acid thereof. In otherembodiments, the αPTHF/taxane-based chemotherapeutic agent complexcomprises an analog of paclitaxel (PTX), or a salt or acid thereof. Insome embodiments, the molar ratio of αPTHF/paclitaxel (or paclitaxelsalt or acid) in the complex is in the range 1-20:1, or any rangetherein between. In further embodiments, the molar ratio ofαPTHF/paclitaxel (or paclitaxel salt or acid) in the complex is in therange 1-10:1, or any range therein between. In further embodiments, themolar ratio of αPTHF/paclitaxel (or paclitaxel salt or acid) in thecomplex is in the range 2-8:1, or any range therein between. In someembodiments, the molar ratio of αPTHF/paclitaxel (or paclitaxel salt oracid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In other embodiments, the molar ratio ofαPTHF/paclitaxel (or paclitaxel salt or acid) in the complex is in therange 1:1-20, 1:1-10, or 1:2-8, or any range therein between. In someembodiments, the molar ratio of αPTHF/paclitaxel (or paclitaxel salt oracid) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12,1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. Inadditional embodiments, the αPTHF/paclitaxel (or paclitaxel salt oracid) complex is encapsulated in a liposome (e.g., as described hereinor otherwise known in the art).

In additional embodiments, the disclosure provides a complex comprisingαPTHF and docetaxel (DTX), or a salt or acid thereof. In otherembodiments, the αPTHF/taxane-based chemotherapeutic agent complexcomprises an analog of docetaxel (DTX), or a salt or acid thereof. Insome embodiments, the molar ratio of αPTHF/docetaxel (or docetaxel saltor acid) in the complex is in the range 1-20:1, or any range thereinbetween. In some embodiments, the molar ratio of αPTHF/docetaxel (ordocetaxel salt or acid) in the complex is in the range 1-10:1, or anyrange therein between. In further embodiments, the molar ratio ofαPTHF/docetaxel (or docetaxel salt or acid) in the complex is in therange 2-8:1, or any range therein between. In some embodiments, themolar ratio of αPTHF/docetaxel (or docetaxel salt or acid) in thecomplex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1,or >50:1. In other embodiments, the molar ratio of αPTHF/docetaxel (ordocetaxel salt or acid) in the complex is in the range 1:1-20, 1:1-10,or 1:2-8, or any range therein between. In some embodiments, the molarratio of αPTHF/docetaxel (or docetaxel salt or acid) is: 1:1, 1:2, 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16,1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments,the αPTHF/docetaxel (or docetaxel salt or acid) complex is encapsulatedin a liposome (e.g., as described herein or otherwise known in the art).

In additional embodiments, the disclosure provides a complex comprisingαPTHF and larotaxel (LTX), or a salt or acid thereof. In otherembodiments, the αPTHF/taxane-based chemotherapeutic agent complexcomprises an analog of larotaxel (LTX), or a salt or acid thereof. Insome embodiments, the molar ratio of αPTHF/larotaxel (or larotaxel saltor acid) in the complex is in the range 1-20:1, or any range thereinbetween. In further embodiments, the molar ratio of αPTHF/larotaxel (orlarotaxel salt or acid) in the complex is in the range 1-10:1, or anyrange therein between. In further embodiments, the molar ratio ofαPTHF/larotaxel (or larotaxel salt or acid) in the complex is in therange 2-8:1, or any range therein between. In some embodiments, themolar ratio of αPTHF/larotaxel (or larotaxel salt or acid) in thecomplex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1,or >50:1. In other embodiments, the molar ratio of αPTHF/larotaxel (orlarotaxel salt or acid) in the complex is in the range 1:1-20, 1:1-10,or 1:2-8, or any range therein between. In some embodiments, the molarratio of αPTHF/larotaxel (or larotaxel salt or acid) is: 1:1, 1:2, 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16,1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments,the αPTHF/larotaxel (or larotaxel salt or acid) complex is encapsulatedin a liposome (e.g., as described herein or otherwise known in the art).

In additional embodiments, the disclosure provides a complex comprisingαPTHF and cabazitaxel (CTX), or a salt or acid thereof. In otherembodiments, the αPTHF/taxane-based chemotherapeutic agent complexcomprises an analog of cabazitaxel (CTX), or a salt or acid thereof. Insome embodiments, the molar ratio of αPTHF/cabazitaxel (or cabazitaxelsalt or acid) in the complex is in the range 1-20:1, or any rangetherein between. In further embodiments, the molar ratio ofαPTHF/cabazitaxel (or cabazitaxel salt or acid) in the complex is in therange 1-10:1, or any range therein between. In further embodiments, themolar ratio of αPTHF/cabazitaxel (or cabazitaxel salt or acid) in thecomplex is in the range 2-8:1, or any range therein between. In someembodiments, the molar ratio of αPTHF/cabazitaxel (or cabazitaxel saltor acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In other embodiments, the molar ratio ofαPTHF/cabazitaxel (or cabazitaxel salt or acid) in the complex is in therange 1:1-20, 1:1-10, or 1:2-8, or any range therein between. In someembodiments, the molar ratio of αPTHF/cabazitaxel (or cabazitaxel saltor acid) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11,1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or1:>50. In additional embodiments, the αPTHF/cabazitaxel (or cabazitaxelsalt or acid) complex is encapsulated in a liposome (e.g., as describedherein or otherwise known in the art).

In additional embodiments, the disclosure provides a complex comprisingαPTHF and another anti-metabolite, or a salt or acid thereof. Ananti-metabolite is a chemical with a structure that is similar to ametabolite required for normal biochemical reactions, yet differentenough to interfere with one or more normal functions of cells, such ascell division. In some embodiments, the disclosure provides a complexcomprising αPTHF and tetrahydrofolate (THF), or a salt or acid thereof.In some embodiments, the disclosure provides a complex comprising αPTHFand an anti-metabolite selected from the group consisting of,gemcitabine, fluorouracil, capecitabine, an antifolate (e.g.,tetrahydrofolate, tetrahydrofolate), tegafur, cytosine arabinoside,thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine,6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, aswell as pharmaceutically acceptable salt or acids, acids, or derivativesof any of these. In some embodiments, the molar ratio ofαPTHF/anti-metabolite (or anti-metabolite salt or acid) in the complexis in the range 1-20:1, or any range therein between. In furtherembodiments, the molar ratio of αPTHF/anti-metabolite (oranti-metabolite salt or acid) in the complex is in the range 1-10:1, orany range therein between. In further embodiments, the molar ratio ofαPTHF/anti-metabolite (or anti-metabolite salt or acid) in the complexis in the range 2-8:1, or any range therein between. In someembodiments, the molar ratio of αPTHF/anti-metabolite (oranti-metabolite salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molarratio of αPTHF/anti-metabolite (or anti-metabolite salt or acid) in thecomplex is in the range 1:1-20, 1:1-10, or 1:2-8, or any range thereinbetween. In some embodiments, the molar ratio of αPTHF/anti-metabolite(or anti-metabolite salt or acid) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7,1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19,1:20, 1:(21-50), or 1:>50. In additional embodiments, theαPTHF/anti-metabolite (or anti-metabolite salt or acid) complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In additional embodiments, the disclosure provides a complex of αPTHF(e.g., a αPTHF disclosed herein) and a cyclodextrin. Cyclodextrins (CDs)are groups of cyclic oligosaccharides which have been shown to improvephysicochemical properties of many drugs through formation of complexes.CDs are cyclic oligosaccharides composed of several D-glucose unitslinked by α-(1,4) bonds. This cyclic configuration provides ahydrophobic internal cavity and gives the CDs a truncated cone shape.Many hydroxyl groups are situated on the edges of the ring which makethe CDs both lipophilic and soluble in water. As a result, CDs are ableto form complexes with a wide variety of hydrophobic agents, and thuschange the physical-chemical properties of these complexed agents.

The terms “cyclodextrin” or “CD” unless otherwise specified herein,refer generally to a parent or derivatized cyclic oligosaccharidecontaining a variable number of (α-1,4)-linked D-glucopyranoside unitsthat is able to form a complex with a tetrahydrofolate-PG. Eachcyclodextrin glucopyranoside subunit has secondary hydroxyl groups atthe 2 and 3 positions and a primary hydroxyl group at the 6-position.The terms “parent”, “underivatized”, or “inert”, cyclodextrin refer to acyclodextrin containing D-glucopyranoside units having the basic formulaC₆H₁₂O₆ and a glucose structure without any additional chemicalsubstitutions (e.g., α-cyclodextrin consisting of 6 D-glucopyranosideunits, a β-cyclodextrin consisting of 7 D-glucopyranoside units, and aα-cyclodextrin consisting of 8 D-glucopyranoside units). The physicaland chemical properties of a parent cyclodextrin can be modified byderivatizing the hydroxyl groups with other functional groups. Anysubstance located within the cyclodextrin internal phase is said to be“complexed” with the cyclodextrin, or to have formed a complex(inclusion complex) with the cyclodextrin.

As used herein, there are no particular limitations on the cyclodextrincomponent of the αPTHF/cyclodextrin complexes so long as thecyclodextrins can form complexes with the αPTHF. In particularembodiments, the cyclodextrins have been derivatized to bear ionizable(e.g., weakly basic and/or weakly acidic) functional groups tofacilitate complex formation with αPTHF and/or liposome encapsulation.

Modifications of the hydroxyl groups of cyclodextrins, such as thosefacing away from the cyclodextrin interior phase, with ionizablechemical groups is known to facilitate the loading of cyclodextrins andtherapeutic agents complexed with the cyclodextrins. In someembodiments, the cyclodextrin of the αPTHF/cyclodextrin complex has atleast 2, 3, 4, 5, 6, 6, 7, 8, 9, or 10, hydroxyl group substituted withan ionizable chemical group. The term “charged cyclodextrin” refers to acyclodextrin having one or more of its hydroxyl groups substituted witha charged moiety. Such a moiety can itself be a charged group or it cancomprise an organic moiety (e.g., a C₁-C₆ alkyl or C₁-C₆ alkyl ethermoiety) substituted with one or more charged moieties.

In some embodiments, the “ionizable” or “charged” moieties of a CDderivative are weakly ionizable. Weakly ionizable moieties are thosethat are either weakly basic or weakly acidic. Weakly basic functionalgroups (W) have a pKa of between about 6.0-9.0, 6.5-8.5, 7.0-8.0,7.5-8.0, and any range in between inclusive according to CH3-W.Similarly, weakly acidic functional groups (X) have a log dissociationconstant (pKa) of between about 3.0-7.0, 4.0-6.5, 4.5-6.5, 5.0-6.0,5.0-5.5, and any range in between inclusive according to CH3-X.Representative anionic moieties include, without limitation,carboxylate, carboxymethyl, succinyl, sulfonyl, phosphate, sulfoalkylether, sulphate carbonate, thiocarbonate, dithiocarbonate, phosphate,phosphonate, sulfonate, nitrate, and borate groups. Representativecationic moieties include, without limitation, amino, guanidine, andquarternary ammonium groups.

In another embodiment, the derivatized cyclodextrin is a “polyanion” or“polycation.” A polyanion is a derivatized cyclodextrin having more thanone negatively charged group resulting in net a negative ionic charge ofmore than two units. A polycation is a derivatized cyclodextrin havingmore than one positively charged group resulting in net positive ioniccharger of more than two units.

In another embodiment, the derivatized cyclodextrin is a “chargeableamphiphile.” By “chargeable” is meant that the amphiphile has a pK inthe range pH 4 to 8, or pH 4 to 8.5. A chargeable amphiphile maytherefore be a weak acid or base. By “amphoteric” herein is meant aderivatized cyclodextrin having a ionizable groups of both anionic andcationic character wherein: (a) at least one, and optionally both, ofthe cation and anionic amphiphiles is chargeable, having at least onecharged group with a pK between 4 and 8 to 8.5, (b) the cationic chargeprevails at pH 4, and (c) the anionic charge prevails at pH 8 to 8.5.

In some embodiments, the “ionizable” or “charged” derivatizedcyclodextrin as a whole, whether polyionic, amphiphilic, or otherwise,are weakly ionizable (e.g., have a pKai of between about 4.0-8.5,4.5-8.0, 5.0-7.5, 5.5-7.0, 6.0-6.5, and any range in between inclusive).

Any one, some, or all hydroxyl groups of any one, some or allα-D-glucopyranoside units of a cyclodextrin can be modified to anionizable chemical group as described herein. Since each cyclodextrinhydroxyl group differs in chemical reactivity, reaction with a modifyingmoiety can produce an amorphous mixture of positional and opticalisomers. Alternatively, certain chemistry can allow for pre-modifiedα-D-glucopyranoside units to be reacted to form uniform products.

The aggregate substitution that occurs for cyclodextrin derivatives in amixture is described by a term referred to as the degree ofsubstitution. For example, a 6-ethylenediamino-β-cyclodextrin with adegree of substitution of seven would be composed of a distribution ofisomers of 6-ethylenediamino-β-cyclodextrin in which the average numberof ethylenediamino groups per 6-ethylenediamino-β-cyclodextrin moleculeis seven. The degree of substitution for a cyclodextrin derivativemixture can routinely be determined using mass spectrometry or nuclearmagnetic resonance spectroscopy.

In one embodiment, at least one hydroxyl moieties facing away from thecyclodextrin interior is substituted with an ionizable chemical group.For example, the C2, C3, C6, C2 and C3, C2 and C6, C3 and C6, and allthree of C2-C3-C6 hydroxyls of at least one α-D-glucopyranoside unit aresubstituted with an ionizable chemical group. Any such combination ofhydroxyls can similarly be combined with at least two, three, four,five, six, seven, eight, nine, ten, eleven, up to all of thealpha-D-glucopyranoside units in the modified cyclodextrin as well as incombination with any degree of substitution described herein. One suchderivative is a sulfoalkyl ether cyclodextrin (SAE-CD). Sulfobutyl etherderivatives of beta cyclodextrin (SBE-β-CD) have been demonstrated tohave significantly improved aqueous solubility compared to the parentcyclodextrin.

Additional cyclodextrin derivatives that may be complexed withtherapeutic agents in the disclosed liposome compositions includesugammadex or Org-25969, in which the 6-hydroxy groups on γ-CD have beenreplaced by carboxythio acetate ether linkages, and hydroxybutenyl-β-CD.Alternative forms of cyclodextrin include: 2,6-Di-O-methyl-β-CD (DIMEB),2-hydroxylpropyl-3-cyclodextrin (HP-β-CD), randomlymethylated-β-cyclodextrin (RAMEB), sulfobutyl ether β-cyclodextrin(SBE-β-CD), and sulfobutylether-γ-cyclodextrin (SBEαCD), sulfobutylatedbeta-cyclodextrin sodium salt, (2-Hydroxypropyl)-alpha-cyclodextrin,(2-Hydroxypropyl)-beta-cyclodextrin, (2-Hydroxypropyl)-γ-cyclodextrin,2,6-di-O-methyl)-beta-cyclodextrin (DIMEB-50 Heptakis),2,3,6-tri-O-methyl)-beta-cyclodextrin (TRIMEB Heptakis),methyl-beta-cyclodextrin, octakis (6-deoxy-6-iodo)-γ-cyclodexrin, and,octakis (6-deoxy-6-bromo)-gamma-cyclodexrin.

In some embodiments, the cyclodextrin(s) has a high solubility in waterin order to facilitate entrapment of a larger amount of the cyclodextrinin the liposome internal phase. In some embodiments, the watersolubility of the cyclodextrin is at least 10 mg/mL, 20 mg/mL, 30 mg/mL,40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL orhigher. In some embodiments, the water solubility of the cyclodextrin(s)is within a range of 10-150 mg/mL, 20-100 mg/mL 20-75 mg/mL, and anyrange in between inclusive.

In some embodiments, a large association constant between thecyclodextrin and the αPTHF and/or other therapeutic agent complexed withcyclodextrin is preferable and can be obtained by selecting the numberof glucose units in the cyclodextrin based on the size of thetherapeutic agent (see, for example, Albers et al., Crit. Rev. Therap.Drug Carrier Syst. 12:311-337 (1995); Stella et al., Toxicol. Pathol.36:30-42 (2008). When the association constant depends on pH, thecyclodextrin can be selected such that the association constant becomeslarge at the pH of the liposome internal phase. As a result, thesolubility (nominal solubility) of the therapeutic agent in the presenceof cyclodextrin can be further improved. In some embodiments, theassociation constant of the cyclodextrin with the therapeutic agent is100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, or higher. In someembodiments, the association constant of the cyclodextrin with thetherapeutic agent is in the range 100-1, 200, 200-1,000, 300-750, andany range therein between.

In some embodiments, the cyclodextrin of the αPTHF/cyclodextrin complexand/or cyclodextrin/therapeutic agent complex is underivatized.

In some embodiments, the cyclodextrin of the αPTHF/cyclodextrin complexand/or cyclodextrin/therapeutic agent complex is derivatized. In furtherembodiments, the cyclodextrin derivative of the complex has thestructure of Formula I:

wherein: n is 4, 5, or 6;wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ are each, independently,—H, a straight chain or branched C₁-C₈-alkylene group, or an optionallysubstituted straight-chain or branched C₁-C₆ group, wherein at least oneof R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branchedC₁-C₈-alkylene (e.g., C₁-C₈-(alkylene)-SO₃ ⁻ group);

In some embodiments, the cyclodextrin derivative of theαPTHF/cyclodextrin complex and/or cyclodextrin/therapeutic agent complexhas the structure of formula II:

-   wherein: n is 4, 5, or 6;-   wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ are each,    independently, —O— or a —O—(C₂-C₆ alkylene)-SO₃— group; wherein at    least one of R₁ and R₂ is independently a —O—(C₂-C₆ alkylene)-SO₃ ⁻    group; and S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ are each,    independently, a pharmaceutically acceptable cation. In further    embodiments, the pharmaceutically acceptable cation is selected    from: an alkali metal such as Li⁺, Na⁺, or K⁺; an alkaline earth    metal such as Ca⁺², or M⁺² and ammonium ions and amine cations such    as the cations of (C1-C6)-alkylamines, piperidine, pyrazine,    (C1-C6)-alkanolamine and (C4-C8)-cycloalkanolamine. In some    embodiments, at least one of R1 and R2 is independently a —O—(C2-C6    alkylene)-SO₃— group that is a —O—(CH₂)_(m)SO3- group, wherein m is    2 to 6, preferably 2 to 4, (e.g., —O—CH2CH2CH2S03- or    —O—CH2CH2CH2CH2S03-); and S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ are    each, independently, H or a pharmaceutically cation which includes    for example, alkali metals (e.g., Li⁺, Na⁺, K⁺) alkaline earth    metals (e.g., Ca⁺², Mg⁺²), ammonium ions and amine cations such as    the cations of (C1-C6)-alkylamines, piperidine, pyrazine,    (C₁-C₆)-alkanol-amine and (C₄-C₈)-cycloalkanolamine:

In some embodiments, a cyclodextrin derivative of the αPTHF/cyclodextrincomplex and/or cyclodextrin/therapeutic agent complex is a cyclodextrindisclosed in U.S. Pat. Nos. 6,133,248, 5,874,418, 6,046,177, 5,376,645,5,134,127, 7,034,013, 6,869,939; and Intl. Appl. Publ. No. WO02005/117911, the contents each of which is herein incorporated byreference in its priority.

In some embodiments, the cyclodextrin derivative of theαPTHF/cyclodextrin complex and/or cyclodextrin/therapeutic agent complexis a sulfoalkyl ether cyclodextrin. In some embodiments, thecyclodextrin derivative of complex is a sulfobutyl ether-3-cyclodextrinsuch as CAPTISOL® (CyDex Pharma. Inc., Lenexa, Kans. Methods forpreparing sulfobutyl ether-3-cyclodextrin and other sulfoalkyl ethercyclodextrins are known in the art.

In some embodiments, the cyclodextrin derivative in of theαPTHF/cyclodextrin complex and/or cyclodextrin/therapeutic agent complexis a compound of Formula III:

wherein R equals:

(a) (H)_(21-X) or (—(CH₂)₄—SO₃Na)_(x), and x=1.0-10.0, 1.0-5.0, 6.0-7.0,or 8.0-10.0;

(b) (H)_(21-X) or (—(CH₂CH(OH)CH₃)x, and x=1.0-10.0, 1.0-5.0, 6.0-7.0,or 8.0-10.0;

(c) (H)_(21-X) or (sulfoalkyl ethers)x, and x=1.0-10.0, 1.0-5.0,6.0-7.0, or 8.0-10.0; or

(d) (H)_(21-X) or (—(CH₂)₄—SO₃Na)x, and x=1.0-10.0, 1.0-5.0, 6.0-7.0, or8.0-10.0.

In additional embodiments, the αPTHF/cyclodextrin complex and/orcyclodextrin/therapeutic agent complex is encapsulated in a liposome(e.g., as described herein or otherwise known in the art).

III. αPTHF Delivery Vehicles

In alternative embodiments, the disclosure provides αPTHF deliverysystems and their use to deliver a payload of αPTHF to a cell or cellsin vitro or in vivo. In some embodiments, αPTHF is complexed with orincorporated into a delivery vehicle. Such delivery vehicles are knownin the art and include, but are not limited to, liposomes, lipospheres,polymers, peptides, proteins, antibodies (e.g., ADCs such asAntibody-αPTHF conjugates), cellular components, cyclic oligosaccharides(e.g., cyclodextrins), nanoparticles (e.g., lipid nanoparticles,biodegradable nanoparticles, and core-shell nanoparticles), lipoproteinparticles, and combinations thereof. In particular embodiments, thedelivery vehicle is a liposome. In other particular embodiments, thedelivery vehicle is an antibody or an antigen binding antibody fragment.

A. Liposomes

In some embodiments, the disclosure provides liposomal compositions thatcomprise a liposome encapsulating (i.e., filled with) an αPTHF (e.g., aαPTHF disclosed herein). In some embodiments, a liposome in theliposomal composition comprises a αPTHF containing 4, 5, 2-10, 4-6, ormore than 5, glutamyl groups (including the glutamyl group intetrahydrofolate). In some embodiments, the αPTHF in the Lp-αPTHFcomprises two or more glutamyl groups in the L-form. In otherembodiments, the αPTHF in the Lp-αPTHF comprises a glutamyl group in theD-form. In further embodiments, the αPTHF in the Lp-αPTHF comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form. In additional embodiments, the αPTHF in the Lp-αPTHF comprisestwo or more glutamyl groups that have a gamma carboxyl linkage. In someembodiments, the polyglutamated alpha tetrahydrofolate in the Lp-αPTHFcomprises at least one glutamyl group that has both an alpha carboxyllinkage and a gamma carboxyl linkage. In some embodiments, the liposomalcomposition comprises a liposome comprising a tetraglutamated THF. Insome embodiments, the liposomal composition comprises a liposomecomprising a pentaglutamated THF. In some embodiments, the liposomalcomposition comprises a liposome comprising a hexaglutamated αPTHF(Lp-αPTHF). In some embodiments, the liposomal composition comprises aliposome that is anionic or neutral. In some embodiments, the liposomalcomposition comprises a liposome that is cationic. In some embodiments,the Lp-αPTHF composition is unpegylated. In some embodiments, theLp-αPTHF composition is non-targeted (NTLp-αPTHF). In other embodiments,the Lp-αPTHF composition is targeted (TLp-αPTHF). In some embodiments,the liposomal composition comprises a liposome having a diameter in therange of 20 nm to 500 nm, or any range therein between. In someembodiments, the liposomal composition comprises a liposome having adiameter in the range of 20 nm to 400 nm, or any range therein between.In some embodiments, the liposomal composition comprises a liposomehaving a diameter in the range of 20 nm to 300 nm or any range thereinbetween. In some embodiments, the liposomal composition comprises aliposome having a diameter in the range of 20 nm to 200 nm, or any rangetherein between. In further embodiments, the liposomal compositioncomprises a liposome having a diameter in the range of 20 nm to 150 nm,or any range therein between. In further embodiments, the liposomalcomposition comprises a liposome having a diameter in the range of 80 nmto 120 nm, or any range therein between. In additional embodiments,30-70%, 30-60%, or 30-50% w/w αPTHF, or any range therein between, isencapsulated (entrapped) in the Lp-αPTHF during the process of preparingthe liposomes. In some embodiments, the Lp-αPTHF composition comprisesat least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, or more than 75%, w/w of the polyglutamated alpha THF. Insome embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75%, w/w, αPTHF, isencapsulated in the Lp-αPTHF during the process of preparing theliposomes.

In some embodiments, the provided liposomes further comprise animmunostimulatory agent, a detectable marker, or both disposed on itsexterior. The immunostimulatory agent or detectable marker can beionically bonded or covalently bonded to an exterior of the liposome,including, for example, optionally to a steric stabilizer component ofthe liposome.

The terms “immunostimulatory agents”, also known as “immunostimulants”,and “immunostimulators”, refer to substances that stimulate an immune(including a preexisting immune response) by inducing activation orincreasing activity of any of the components of the immune system. Theseimmunostimulatory agents can include one or more of a hapten, anadjuvant, a protein immunostimulating agent, a nucleic acidimmunostimulating agent, and a chemical immunostimulating agent. Manyadjuvants contain a substance designed to stimulate immune responses,such as lipid A, Bortadella pertussis or Mycobacterium tuberculosisderived proteins. Certain adjuvants are commercially available as, forexample, Freund's Incomplete Adjuvant and Complete Adjuvant (DifcoLaboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company,Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.);aluminum salts such as aluminum hydroxide gel (alum) or aluminumphosphate; salts of calcium, iron or zinc; an insoluble suspension ofacylated tyrosine; acylated sugars; cationically or anionicallyderivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A; IFN-alpha, IFN-alpha,FLT3-ligand; and immunostimulatory antibodies (e.g., anti-CTLA-4,anti-CD28, anti-CD3. Cytokines, such as GM-CSF, interleukin-2, -7, -12,and -15, and other like growth factors, can also be used as adjuvants.In a preferred embodiment, the immunostimulant can be at least oneselected from the group consisting of fluorescein, DNP, beta glucan,beta-1,3-glucan, beta-1,6-glucan. In an additional preferred embodiment,the immunostimulant is a Toll-like receptor (TLR) modulating agent. Infurther embodiments, the Toll-like receptor (TLR) modulating agent isone or more of: an oxidized low-density lipoprotein (e.g., OXPAC, PGPC),an eritoran lipid (e.g., E5564), and a Resolvin. In some embodiments,the liposomes comprise fluorescein isothiocyanate (FITC) which, based onour experiments, surprisingly serves as both an immunostimulant and adetectable marker.

In some embodiments, the liposomes comprise a detectable marker. Adetectable marker may, for example, include, at least, a radioisotope, afluorescent compound, a bioluminescent compound, chemiluminescentcompound, a metal chelator, an enzyme, a dye, an ink, a magneticcompound, a biocatalyst or a pigment that is detectable by any suitablemeans known in the art, e.g., magnetic resonance imaging (MRI), opticalimaging, fluorescent/luminescent imaging, and/or nuclear imagingtechniques.

In some embodiments, the immunostimulatory agent and/or detectablemarker is attached to the exterior by co-incubating it with theliposome. For example, the immunostimulatory agent and/or detectablemarker may be associated with the liposomal membrane by hydrophobicinteractions or by an ionic bond such as an avidin/biotin bond or ametal chelation bond (e.g., Ni-NTA). Alternatively, theimmunostimulatory agent or detectable marker may be covalently bonded tothe exterior of the liposome such as, for example, by being covalentlybonded to a liposomal component or to the steric stabilizer which is thePEG.

In some embodiments, the liposomes further comprise an agent thatincreases the uptake of liposomes into a cellular compartment ofinterest including the cytosol.

In some embodiments, the liposomes comprise a mitochondrial-targetingagent. In some embodiments, the liposomes comprise triphenylphosphonium(TPP). Methods and mechanisms for surface functionalizing liposomes withTPP are known in the art (e.g., attaching TPP to the lipid anchor via apeg spacer group and modifying TPP with a stearyl group (stearyltriphenylphosphonium (STPP)). In some embodiments, the liposomescomprise high-density octa-arginine. In some embodiments, the liposomescomprise sphingomyelin and/or a sphingomyelin metabolite. Sphingomyelinmetabolite used to formulate the liposomes of the present invention caninclude, for example ceramide, sphingosine or sphingosine 1-phosphate.In some embodiments, the liposomes comprise Rhodamine 123. In someembodiments, the liposomes comprise, a mitochondria penetrating peptide.In some embodiments, the liposomes comprise, a mitochondria penetratingagent selected from the group consisting of: a mitofusin peptide, amitochondrial targeting signal peptide, and Antennapedia helix IIIhomeodomain cell-penetrating peptide (ANT) (e.g., comprisingRQIKIWFQNRRMKWKKRKKRRQRRR (SEQ ID NO:1), RKKRRXR RRGC where X is anynatural or non-natural amino acid (SEQ ID NO:2), CCGCCAAGAAGCG (SEQ IDNO:3), GCGTGCACACGCGCGTAGACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTGAGCGGCGTGGCGCGGGG GC GTCAT (SEQ IDNO:4), ACGTGCATACGCACGTAGACATTCCCCGCTTCCCACTCCAAAGTCCGCCAAGAAGCGTATCCCGCTGAGCGGCGTGGCGCGGGGGCGTC AT CCGTCAGCTC (SEQ IDNO:5), or ACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAA GAAGCGACCCCTCCGGGGCGAGCTG(SEQ ID NO:6)), or a mitochondrial penetrating fragment thereof.

In some embodiments, liposomes in the provided liposome compositionscomprise a mitochondria penetrating agent selected from the group: aguanidine-rich peptoid, tetraguanidinium, triguanidinium, diguanidinium,monoguanidinium, a guanidine-rich polycarbamate, a beta-oligoarginine, aproline-rich dendrimer, and a phosphonium salt (e.g.,methyltriphenyl-phosphonium and/or tetraphenylphosphonium).

In some embodiments, liposomes in the provided liposome compositionscomprise sphingomyelin and/or stearyl-octa-arginine. In someembodiments, the liposomes comprise sphingomyelin and/orstearyl-octa-arginine. In some embodiments, the liposomes comprise DOPE,sphingomyelin, stearyl-octa-arginine sphingomyelin andstearyl-octa-arginine. In some embodiments, the liposomes comprise DOPE,sphingomyelin, stearyl-octa-arginine sphingomyelin andstearyl-octa-arginine at a molar ratio of 9:2:1. In some embodiments,the liposomes comprise the MITO-porter® system or a variant thereof.

In some embodiments, liposomes in the provided liposome compositionscomprise an agent such as a cell penetrating agent that that facilitatesdelivery of the liposome across a cell membrane and provides theliposome with the ability to bypass the endocytic pathway and the harshenvironment of lysosomes. Cell penetrating agents are known in the artand can routinely be used and adapted for manufacture and use of theprovided liposome compositions. In some embodiments, the cellpenetrating/lysosome bypassing agent is chloroquine. In someembodiments, the cell penetrating agent is a cell penetrating peptide.In some embodiments, liposomes in the provided liposome compositionscomprise a cell penetrating agent selected from the group: RKKRRQRRR(SEQ ID NO:7), GRKKRRQRRRTPQ (SEQ ID NO:8), YGRKKRRQRRR (SEQ ID NO:9),AAVAL LPAVLLALLA (SEQ ID NO:10), MGLGLHLLVLAAALQ (SEQ ID NO:11), GALFLGFLGAAGSTM (SEQ ID NO:12), AGYLLGKINLKALAALAKKIL (SEQ ID NO:13),RVIRVWFQNKRCKDKK (SEQ ID NO:14), RQIKIWFQNRRMKWKK (SEQ ID NO:15),GLFEAIAGFIENGWEGMIDG (SEQ ID NO:16), GWTLNSAGYLLGKIN (SEQ ID NO:17),RSQSRSRYYRQRQRS (SEQ ID NO:18), LAIPEQEY (SEQ ID NO:19), LGIAEQEY (SEQID NO:20), LGIPAQEY (SEQ ID NO:21), LGIPEAEY (SEQ ID NO:22), LGIPEQAY(SEQ ID NO:23), LGIAEAEY (SEQ ID NO:24), LGIPEAAY (SEQ ID NO:25),LGIAEQAY (SEQ ID NO:26), LGIAEAAY (SEQ ID NO:27), LLIILRRRIRKQAHAHSK(SEQ ID NO:28), LKALAALAKKIL (SEQ ID NO:29), KLALKLALKALKAALKLA (SEQ IDNO:30), KETWWETWWTEWSQPKKKRKV (SEQ ID NO:31), DHQLNPAF (SEQ ID NO:32),DPKGDPKG (SEQ ID NO:33), VTVTVTVTVTGKGDPKPD (SEQ ID NO:34),RQIKIWFQNRRMKWKK (SEQ ID NO:35), GRKKRRQRRRPPQ (SEQ ID NO:36),GWTLNSAGYLLGKINLKALAAL AKKIL (SEQ ID NO:37), GRKKRRQRRR (SEQ ID NO:38),RRRRRRR (SEQ ID NO:39), RRRRRRRR (SEQ ID NO:40), RRRRRRRRR (SEQ IDNO:41), RRRRRRRR RR (SEQ ID NO:42), RRRRRRRRRRR (SEQ ID NO:43), andYTIWMPENPRPGT PCDIFTNSRGKRASNGGG G(R)n wherein n=2-15 R in the L- and/orD-form (SEQ ID NO:44), or a cell permeating fragment thereof.

As discussed above, the liposomes may comprise a steric stabilizer thatcan increase their longevity in circulation. For those embodiments,which incorporate a steric stabilizer, the steric stabilizer may be atleast one member selected from the group consisting of polyethyleneglycol (PEG), poly-L-lysine (PLL), monosialoganglioside (GM1),poly(vinyl pyrrolidone) (PVP), poly(acrylamide) (PAA),poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), phosphatidylpolyglycerol, poly[N-(2-hydroxypropyl) methacrylamide], amphiphilicpoly-N-vinylpyrrolidones, L-amino-acid-based polymer, oligoglycerol,copolymer containing polyethylene glycol and polypropylene oxide,Poloxamer 188, and polyvinyl alcohol. In some embodiments, the stericstabilizer or the population of steric stabilizer is PEG. In oneembodiment, the steric stabilizer is a PEG. In a further embodiment, thePEG has a number average molecular weight (Mn) of 200 to 5000 daltons.These PEG(s) can be of any structure such as linear, branched, star orcomb structure and are commercially available.

In some embodiments, the liposomal composition comprises a pegylatedliposome (PLp-αPTHF). In some embodiments, a pegylated liposome in theliposomal composition comprises a αPTHF containing 4, 5, 2-10, 4-6, ormore than 5, glutamyl groups. In some embodiments, the αPTHF in theLp-αPTHF comprises two or more glutamyl groups in the L-form. In otherembodiments, the αPTHF in the Lp-αPTHF comprises a glutamyl group in theD-form. In further embodiments, the αPTHF in the Lp-αPTHF comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form. In additional embodiments, the polyglutamated alphatetrahydrofolate in the Lp-αPTHF comprises two or more glutamyl groupsthat have an alpha linkage. In some embodiments, at least one glutamylgroup has both an alpha linkage and a gamma linkage. In someembodiments, the liposomal composition comprises a pegylated liposomecomprising a pentaglutamated THF. In further embodiments, the liposomecomprises an L-pentaglutamated THF, a D-pentaglutamated THF, or an L-and D-pentaglutamated THF. In some embodiments, the liposomalcomposition comprises a pegylated liposome comprising a hexaglutamatedTHF. In further embodiments, the liposome comprises an L-hexaglutamatedαPTHF, a D-hexaglutamated THF, or an L- and D-hexaglutamated THF. Insome embodiments, the liposomal composition comprises a pegylatedliposome that is anionic or neutral. In some embodiments, the liposomalcomposition comprises a pegylated liposome that is cationic. In someembodiments, the PLp-αPTHF composition is non-targeted (NTPLp-αPTHF). Inother embodiments, the PLp-αPTHF composition is targeted (TPLp-αPTHF).In some embodiments, the liposomal composition comprises at least 1%,5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,or more than 75%, w/w of the αPTHF. In some embodiments, the liposomalcomposition comprises a pegylated liposome comprising at least 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, ormore than 75%, w/w of the αPTHF is encapsulated (entrapped) in thePLp-αPTHF during the process of preparing the liposomes. In someembodiments, the liposomal composition comprises a pegylated liposomehaving a diameter in the range of 20 nm to 500 nm. In some embodiments,the liposomal composition comprises a pegylated liposome having adiameter in the range of 20 nm to 400 nm. In some embodiments, theliposomal composition comprises a pegylated liposome having a diameterin the range of 20 nm to 300 nm. In some embodiments, the liposomalcomposition comprises a pegylated liposome having a diameter in therange of 20 nm to 200 nm. In further embodiments, the liposomalcomposition comprises a pegylated liposome having a diameter in therange of 80 nm to 120 nm.

In some embodiments, greater than 70%, 80% or 90% of the polyglutamatedtetrahydrofolate in a provided liposomal composition is pentaglutamated.In some embodiments, greater than 70%, 80% or 90% of the polyglutamatedtetrahydrofolate in a provided composition is hexaglutamated. In someembodiments, greater than 70%, 80% or 90% of the polyglutamatedtetrahydrofolate in the composition has 4-10, 4-6, or more than 5,glutamyl groups.

In some embodiments, the αPTHF compositions (e.g., polyglutamates anddelivery vehicles such as liposomes containing the polyglutamates) arein an aqueous solution. In some embodiments, the αPTHF composition isadministered in a liposomal composition at a dose of between 0.005 and5000 mg of αPTHF per square meter (m²) of body surface area, or anyrange therein between. In further embodiments, the αPTHF composition isadministered in a liposomal composition at a dose of between 0.1 and1000 mg αPTHF per square meter of body surface area, or any rangetherein between.

(1) Liposome Composition

The lipids and other components of the liposomes contained in theliposomal compositions can be any lipid, lipid combination and ratio, orcombination of lipids and other liposome components and their respectiveratios known in the art. However, it will be understood by one skilledin the art that liposomal encapsulation of any particular drug, such as,and without limitation, the polyglutamated alpha THF discussed herein,may involve substantial routine experimentation to achieve a useful andfunctional liposomal formulation. In general, the provided liposomes mayhave any liposome structure, e.g., structures having an inner spacesequestered from the outer medium by one or more lipid bilayers, or anymicrocapsule that has a semi-permeable membrane with a lipophiliccentral part where the membrane sequesters an interior. The lipidbilayer can be any arrangement of amphiphilic molecules characterized bya hydrophilic part (hydrophilic moiety) and a hydrophobic part(hydrophobic moiety). Usually amphiphilic molecules in a bilayer arearranged into two dimensional sheets in which hydrophobic moieties areoriented inward the sheet while hydrophilic moieties are orientedoutward. Amphiphilic molecules forming the provided liposomes can be anyknown or later discovered amphiphilic molecules, e.g., lipids ofsynthetic or natural origin or biocompatible lipids. The liposomes canalso be formed by amphiphilic polymers and surfactants, e.g.,polymerosomes and niosomes. For the purpose of this disclosure, withoutlimitation, these liposome-forming materials also are referred to as“lipids”.

The liposome composition formulations provided herein can be in liquidor dry form such as a dry powder or dry cake. The dry powder or dry cakemay have undergone primary drying under, for example, lyophilizationconditions or optionally, the dry cake or dry powder may have undergoneboth primary drying only or both primary drying and secondary drying. Inthe dry form, the powder or cake may, for example, have between 1% to 6%moisture, for example, such as between 2% to 5% moisture or between 2%to 4% moisture. One example method of drying is lyophilization (alsocalled freeze-drying, or cyrodessication). Any of the compositions andmethods of the disclosure may include liposomes, lyophilized liposomesor liposomes reconstituted from lyophilized liposomes. In someembodiments, the disclosed compositions and methods include one or morelyoprotectants or cryoprotectants. These protectants are typicallypolyhydroxy compounds such as sugars (mono-, di-, and polysaccharides),polyalcohols, and their derivatives, glycerol, or polyethyleneglycol,trehalose, maltose, sucrose, glucose, lactose, dextran, glycerol, oraminoglycosides. In further embodiments, the lyoprotectants orcryoprotectants comprise up to 10% or up to 20% of a solution outsidethe liposome, inside the liposome, or both outside and inside theliposome.

In some embodiments, the liposomes include a steric stabilizer thatincreases their longevity in circulation. One or more steric stabilizerssuch as a hydrophilic polymer (Polyethylene glycol (PEG)), a glycolipid(monosialoganglioside (GM1)) or others occupies the space immediatelyadjacent to the liposome surface and excludes other macromolecules fromthis space. Consequently, access and binding of blood plasma opsonins tothe liposome surface are hindered, and thus interactions of macrophageswith such liposomes, or any other clearing mechanism, are inhibited andlongevity of the liposome in circulation is enhanced. In someembodiments, the steric stabilizer or the population of stericstabilizers is a PEG or a combination comprising PEG. In furtherembodiments, the steric stabilizer is a PEG or a combination comprisingPEG with a number average molecular weight (Mn) of 200 to 5000 daltons.These PEG(s) can be of any structure such as linear, branched, star orcomb structure and are commercially available.

The diameter of the disclosed liposomes is not particularly limited. Insome embodiments, the liposomes have a diameter in the range of forexample, 30-150 nm (nanometer). In other embodiments, the liposomes havea diameter in the range of 40-70 nm.

The properties of liposomes are influenced by the nature of lipids usedto make the liposomes. A wide variety of lipids have been used to makeliposomes. These include cationic, anionic and neutral lipids. In someembodiments, the liposomes comprising the αPTHF are anionic or neutral.In other embodiments, the provided liposomes are cationic. Thedetermination of the charge (e.g., anionic, neutral or cationic) canroutinely be determined by measuring the zeta potential of the liposome.The zeta potential of the liposome can be positive, zero or negative. Insome embodiments, the zeta potential of the liposome is less than orequal to zero. In some embodiments, the zeta potential of the liposomeis in a range of 0 to −150 mV. In another embodiment, the zeta potentialof the liposome is in the range of −30 to −50 mV.

In some embodiments, cationic lipids are used to make cationic liposomeswhich are commonly used as gene transfection agents. The positive chargeon cationic liposomes enables interaction with the negative charge oncell surfaces. Following binding of the cationic liposomes to the cell,the liposome is transported inside the cell through endocytosis.

In some preferred embodiments, a neutral to anionic liposome is used. Ina preferred embodiment, an anionic liposome is used. Using a mixture of,for example, neutral lipids such as HSPC and anionic lipids such asPEG-DSPE results in the formation of anionic liposomes which are lesslikely to non-specifically bind to normal cells. Specific binding totumor cells can be achieved by using a tumor targeting antibody such as,for example, a folate receptor antibody, including, for example, folatereceptor alpha antibody, folate receptor beta antibody and/or folatereceptor delta antibody.

As an example, at least one (or some) of the lipids is/are amphipathiclipids, defined as having a hydrophilic and a hydrophobic portions(typically a hydrophilic head and a hydrophobic tail). The hydrophobicportion typically orients into a hydrophobic phase (e.g., within thebilayer), while the hydrophilic portion typically orients toward theaqueous phase (e.g., outside the bilayer). The hydrophilic portion cancomprise polar or charged groups such as carbohydrates, phosphate,carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxy and other likegroups. The hydrophobic portion can comprise apolar groups that includewithout limitation long chain saturated and unsaturated aliphatichydrocarbon groups and groups substituted by one or more aromatic,cyclo-aliphatic or heterocyclic group(s). Examples of amphipathiccompounds include, but are not limited to, phospholipids, aminolipidsand sphingolipids.

Typically, for example, the lipids are phospholipids. Phospholipidsinclude without limitation phosphatidylcholine,phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol,phosphatidylserine, and the like. It is to be understood that otherlipid membrane components, such as cholesterol, sphingomyelin, andcardiolipin, can be used.

The lipids comprising the liposomes provided herein can be anionic andneutral (including zwitterionic and polar) lipids including anionic andneutral phospholipids. Neutral lipids exist in an uncharged or neutralzwitterionic form at a selected pH. At physiological pH, such lipidsinclude, for example, dioleoylphosphatidylglycerol (DOPG),diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols.Examples of zwitterionic lipids include without limitationdioleoylphosphatidylcholine (DOPC), dimyristoylphos-phatidylcholine(DMPC), and dioleoylphosphatidylserine (DOPS). Anionic lipids arenegatively charged at physiological pH. These lipids include withoutlimitation phosphatidylglycerol, cardiolipin, diacylphosphatidylserine,diacylphosphatidic acid, N-dode-canoyl phosphatidylethanolamines,N-succinyl phosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphos-phatidylglycerol (POPG), and other anionic modifyinggroups joined to neutral lipids.

Collectively, anionic and neutral lipids are referred to herein asnon-cationic lipids. Such lipids may contain phosphorus but they are notso limited. Examples of non-cationic lipids include lecithin,lysolecithin, phosphatidylethanolamine, lysophosphatidylethan-olamine,dioleoylphosphati-dylethanolamine (DOPE), dipalmitoyl phosphatidylethanol-amine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidy 1-ethan-olamine (DSPE),palmitoyloleoyl-phosphatidylethanolamine (POPE)palmitoyl-oleoyl-phosphatidylcholine (POPC), egg phosphatidylcholine(EPC), di stearoylphosphatidyl-choline (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphospha-tidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphospha-tidylglycerol (DPPG),palmitoyloleyolphosphatidylglycerol (POPG), 16-0-monomethyl PE,16-0-dimethyl PE, 18-1-trans PE,palmitoyloleoyl-phosphatidylethanolamine (POPE),1-stearoyl-2-oleoylphospha-tidyethanolamine (SOPE), phosphatidylserine,phosphatidyl-inositol, sphingomyelin, cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetyl-phosphate, and cholesterol.

The liposomes may be assembled using any liposomal assembly method usingliposomal components (also referred to as liposome components) known inthe art. Liposomal components include, for example, lipids such as DSPE,HSPC, cholesterol and derivatives of these components. Other suitablelipids are commercially available for example, by Avanti Polar Lipids,Inc. (Alabaster, Ala., USA). A partial listing of available negativelyor neutrally charged lipids suitable for making anionic liposomes, canbe, for example, at least one of the following: DLPC, DMPC, DPPC, DSPC,DOPC, DMPE, DPPE, DOPE, DMPA.Na, DPPA.Na, DOPA.Na, DMPG.Na, DPPG.Na,DOPG.Na, DMPS.Na, DPPS.Na, DOPS.Na, DOPE-Glutaryl.(Na)2, TetramyristoylCardiolipin .(Na)2, DSPE-mPEG-2000.Na, DSPE-mPEG-5000.Na, andDSPE-Maleimide PEG-2000.Na.

In some embodiments, the αPTHF compositions provided herein areformulated in a liposome comprising a cationic lipid. In one embodiment,the cationic lipid is selected from, but not limited to, a cationiclipid described in Intl. Appl. Publ. Nos. WO2012/040184, WO2011/153120,WO2011/149733, WO2011/090965, WO2011/043913, WO2011/022460,WO2012/061259, WO2012/054365, WO2012/044638, WO2010/080724, WO2010/21865and WO2008/103276, U.S. Pat. Nos. 7,893,302, 7,404,969 and 8,283,333 andUS Appl. Publ. Nos. US20100036115 and US20120202871; each of which isherein incorporated by reference in their entirety. In anotherembodiment, the cationic lipid may be selected from, but not limited to,formula A described in Intl. Appl. Publ. Nos. WO2012/040184,WO2011/153120, WO201/1149733, WO2011/090965, WO2011/043913,WO2011/022460, WO2012/061259, WO2012/054365 and WO2012/044638; each ofwhich is herein incorporated by reference in their entirety. In yetanother embodiment, the cationic lipid may be selected from, but notlimited to, formula CLI-CLXXIX of International Publication No.WO2008103276, formula CLI-CLXXIX of U.S. Pat. No. 7,893,302, formulaCLI-CLXXXXII of U.S. Pat. No. 7,404,969 and formula I-VI of US PatentPublication No. 0520100036115; each of which is herein incorporated byreference in their entirety. As a non-limiting example, the cationiclipid may be selected from(20Z,23Z)—N,N-dimethylnonacosa-20,23-dien-10-amine,(17Z,20Z)—N,N-dimemyl-hexacosa-17,20-dien-9-amine,(1Z,19Z)—N5N-dimethylpentacosa-16, 19-dien-8-amine,(13Z,16Z)—N,N-dimethyl-docosa-13,16-dien-5-amine,(12Z,15Z)—N,N-dimethylhenicosa-12,15-dien-4-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-6-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-7-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-10-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-5-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-4-amine,(19Z,22Z)—N,N-dimeihyloctacosa-19,22-dien-9-amine,(18Z,21Z)—N,N-dimethyl-heptacosa-18,21-dien-8-amine,(17Z,20Z)—N,N-dimethylhexacosa-17,20-dien-7-amine,(16Z,19Z)—N,N-dimethylpentacosa-16,19-dien-6-amine,(22Z,25Z)—N,N-dimethyl-hentriaconta-22,25-dien-10-amine, (21Z,24Z)—N,N-dimethyl-triaconta-21,24-dien-9-amine,(18Z)—N,N-dimetylheptacos-18-en-10-amine,(17Z)—N,N-dimethylhexacos-17-en-9-amine,(19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-7-amine,N,N-dimethylheptacosan-10-amine,(20Z,23Z)—N-ethyl-N-methylnonacosa-20,23-dien-10-amine,1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine,(20Z)—N,N-dimethylheptacos-20-en-1 0-amine, (15Z)—N,N-dimethyleptacos-15-en-1 0-amine, (14Z)—N,N-dimethylnonacos-14-en-10-amine,(17Z)—N,N-dimethylnonacos-17-en-10-amine,(24Z)—N,N-dimethyltritriacont-24-en-10-amine,(20Z)—N,N-dimethylnonacos-20-en-10-amine,(22Z)—N,N-dimethylhentriacont-22-en-10-amine,(16Z)—N,N-dimethylpenta-cos-16-en-8-amine,(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl] eptadecan-8-amine,1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethyl nonadecan-10-amine,N,N-dimethyl-1-[(1 S,2R)-2-octylcyclopropyl]nonadecan-10-amine,N,N-dimethyl-21-[R1S,2R)-2-octylcyclopropyl]-henicosan-10-amine,N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine,N,N-dimethyl-3-{7-[(1S, 2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine,1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine,1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethyl-penta-decan-6-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine,R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propa-n-2-amine,S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine,(2S)—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z-)-oct-5-en-1-yloxy]propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine,(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-ylo-xy]propan-2-amine,(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]pr-opan-2-amine,N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy) propan-2-amine;(2S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine,(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)pro pan-2-amine,(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl1-3-(octyloxy) propan-2-amine,1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dime-thylpropan-2-amine,(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy) propan-2-amine,(2R)—N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octa-deca-9,12-dien-1-yloxy]propan-2-amine,(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-R9Z,12Z)-octadeca-9,12-die-n-1-yloxylpropan-2-amine,N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]-methyl} cyclopropyl] octyl} oxy) propan-2-amine,N,N-dimethyl-1-{[-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and(11E,20Z,23Z)—N,N-dimethylnonacosa-11,20,2-trien-10-amine or apharmaceutically acceptable salt or acid or stereoisomer thereof.

In one embodiment, the lipid may be a cleavable lipid such as thosedescribed in in Intl. Publ. No. WO2012/170889, which is hereinincorporated by reference in its entirety

The cationic lipid can routinely be synthesized using methods known inthe art and/or as described in Intl. Publ. Nos. WO2012/040184,WO2011/153120, WO2011/149733, WO2011/090965, WO201/1043913,WO2011/022460, WO2012/061259, WO2012/054365, WO2012/044638,WO2010/080724 and WO2010/21865; each of which is herein incorporated byreference in its entirety.

Lipid derivatives can include, for example, at least, the bonding(preferably covalent bonding) of one or more steric stabilizers and/orfunctional groups to the liposomal component after which the stericstabilizers and/or functional groups should be considered part of theliposomal components. Functional groups comprises groups that can beused to attach a liposomal component to another moiety such as aprotein. Such functional groups include, at least, maleimide. Thesesteric stabilizers include at least one from the group consisting of:polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside(GM1); poly(vinyl pyrrolidone) (PVP); poly(acrylamide) (PAA);poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidylpolyglycerol; poly[N-(2-hydroxy-propyl) methacrylamide]; amphiphilicpoly-N-vinylpyrrolidones; L-amino-acid-based polymer; and polyvinylalcohol.

In some embodiments, the αPTHF compositions are formulated in alipid-polycation complex. The formation of the lipid-polycation complexmay be accomplished using methods known in the art and/or as describedin U.S. Pub. No. 20120178702, herein incorporated by reference in itsentirety. As a non-limiting example, the polycation may include acationic peptide or a polypeptide such as, but not limited to,polylysine, polyornithine and/or polyarginine and the cationic peptidesdescribed in International Pub. No. WO2012/013326; herein incorporatedby reference in its entirety. In another embodiment, the αPTHF isformulated in a lipid-polycation complex which further includes aneutral lipid such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

Since the components of a liposome can include any molecule(s) (e.g.,chemical/reagent/protein) that is bound to it, in some embodiments, thecomponents of the provided liposomes include, at least, a memberselected from the group DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG;HSPC-maleimide; cholesterol; cholesterol-PEG; and cholesterol-maleimide.In some embodiments, the components of the provided liposomes includeDSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC-maleimide;cholesterol; cholesterol-PEG; and cholesterol-maleimide. In a preferredembodiment, the liposomal components that make up the liposome comprisesDSPE; DSPE-FITC; DSPE-maleimide; cholesterol; and HSPC.

In additional embodiments, the liposomes of the liposome compositionsprovided herein comprise oxidized phospholipids. In some embodiments,the liposomes comprise an oxidize phospholipid of a member selected fromthe group consisting of phosphatidylserines, phosphatidylinositols,phosphatidylethanolamines, phosphatidyl-cholines and1-palmytoyl-2-arachidonoyl-sn-glycero-2-phosphate. In some embodiments,the phospholipids have unsaturated bonds. In some embodiments, thephospholipids are arachidonic acid containing phospholipids. Inadditional embodiments, the phospholipids are sn-2-oxygenated. Inadditional embodiments, the phospholipids are not fragmented.

In some embodiments, the liposomes of the disclosed liposomecompositions comprise oxidized1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC). Theterm “oxPAPC”, as used herein, refers to lipids generated by theoxidation of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine(PAPC), which results in a mixture of oxidized phospholipids containingeither fragmented or full length oxygenated sn-2 residues.Well-characterized oxidatively fragmented species contain a five-carbonsn-2 residue bearing omega-aldehyde or omega-carboxyl groups. Oxidationof arachidonic acid residue also produces phospholipids containingesterified isoprostanes. OxPAPC includes HOdiA-PC, KOdiA-PC, HOOA-PC andKOOA-PC species, among other oxidized products present in oxPAPC. Infurther embodiments, the oxPAPCs are epoxyisoprostane-containingphospholipids. In further embodiments, the oxPAPC is1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine(5,6-PEIPC),1-palmitoyl-2-(epoxy-cyclo-pentenone)-sn-glycero-3-phosphorylcholine(PECPC) and/or 1-palmitoyl-2-(epoxy-isoprostaneE2)-sn-glycero-4-phosphocholine (PEIPC). In some embodiments, thephospholipids have unsaturated bonds. In some embodiments, thephospholipids are arachidonic acid containing phospholipids. Inadditional embodiments, the phospholipids are sn-2-oxygenated. Inadditional embodiments, the phospholipids are not fragmented.

In some embodiments, the liposomal αPTHF composition is pegylated (i.e.,a pegylated liposomal polyglutamated alpha (e.g., pentaglutamated orhexaglutamated) antifolate (PLp-αPTHF or TPLp-αPTHF)). In someembodiments, the PLp-αPTHF or TPLp-αPTHF is water soluble. That is, thePLp-αPTHF or TPLp-αPTHF is in the form an aqueous solution.

In some embodiments, the liposomes of the disclosed liposomecompositions comprise a lipid selected from:1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC);1-palmitoyl-2-(9′oxo-nonanoyl)-sn-glycero-3-phosphocholine;1-palmitoyl-2-arachinodoyl-sn-glycero-3-phosphocholine;1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine;1-palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine;1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine; and1-palmitoyl-2-acetoyl-sn-glycero-3-phospho-choline. In furtherembodiments, the liposome comprises PGPC.

In some embodiments, the pH of solutions comprising the liposomecomposition is from pH 5 to 8 or from pH 2 to 6. 2 to 8, or any rangetherein between. In some embodiments, the pH of solutions comprising theliposome composition is from pH 5 to 8, or any range therein between. Insome embodiments, the pH of solutions comprising the liposomecomposition is from pH 6 to 7, or any range therein between. In someembodiments, the pH of solutions comprising the liposome composition isfrom 6 to 7.5, from 6.5 to 7.5, from 6.7 to 7.5, or from 6.3 to 7.0, orany range therein between.

In some embodiments, at least one component of the liposome lipidbilayer is functionalized (or reactive). As used herein, afunctionalized component is a component that comprises a reactive groupthat can be used to crosslink reagents and moieties to the lipid. If thelipid is functionalized, any liposome that it forms is alsofunctionalized. In some embodiments, the reactive group is one that willreact with a crosslinker (or other moiety) to form crosslinks. Thereactive group in the liposome lipid bilayer is located anywhere on thelipid that allows it to contact a crosslinker and be crosslinked toanother moiety (e.g., a steric stabilizer or targeting moiety). In someembodiments, the reactive group is in the head group of the lipid,including for example a phospholipid. In some embodiments, the reactivegroup is a maleimide group. Maleimide groups can be crosslinked to eachother in the presence of dithiol crosslinkers including but not limitedto dithiolthrietol (DTT).

It is to be understood that the use of other functionalized lipids,other reactive groups, and other crosslinkers beyond those describedabove is further contemplated. In addition to the maleimide groups,other examples of contemplated reactive groups include but are notlimited to other thiol reactive groups, amino groups such as primary andsecondary amines, carboxyl groups, hydroxyl groups, aldehyde groups,alkyne groups, azide groups, carbonyls, halo acetyl (e.g., iodoacetyl)groups, imidoester groups, N-hydroxysuccinimide esters, sulfhydrylgroups, and pyridyl disulfide groups.

Functionalized and non-functionalized lipids are available fromcommercial sources including Avanti Polar Lipids (Alabaster, Ala.) andLipoid LLC (Newark, N.J.).

(2) Liposome Interior Space

In further non-limiting embodiments, the provided liposomes enclose aninterior space. In some embodiments, the interior space comprises, butis not limited to, an aqueous solution. In some embodiments, theinterior space comprises a αPTHF as provided herein. In additionalembodiments, the interior space of the liposome comprises a tonicityagent. In some embodiments. In some embodiments, the concentration(weight percent) of the tonicity agent is 0.1-20%, 1-20%, 0.5-15%,1-15%, or 1-50%, or any range therein between. In some embodiments, theinterior space of the liposome includes a sugar (e.g., trehalose,maltose, sucrose, lactose, mannose, mannitol, glycerol, dextrose,fructose, etc.). In further embodiments, the concentration (weightpercent) of the sugar is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or 1-50%, orany range therein between. In some embodiments, the pH of the interiorspace of the liposome is from pH 2 to 8, or any range therein between.In some embodiments, the pH of solutions comprising the liposomecomposition is from pH 5 to 8, or any range therein between. In someembodiments, the pH of solutions comprising the liposome composition isfrom pH 6 to 7, or any range therein between. In some embodiments, thepH of solutions comprising the liposome composition is from 6 to 7.5,from 6.5 to 7.5, from 6.7 to 7.5, or from 6.3 to 7.0, or any rangetherein between. In some embodiments, the interior space comprisesbuffer. In further embodiments, the buffer a buffer selected from HEPES,citrate, or sodium phosphate (e.g., monobasic and/or dibasic sodiumphosphate). In some embodiments, the buffer is HEPES. In someembodiments, the buffer is citrate. In some embodiments, the buffer issodium phosphate (e.g., monobasic and/or dibasic sodium phosphate). Insome embodiments, the buffer is at a concentration of 15 to 200 mM, orany range therein between. In yet further embodiments, the buffer is ata concentration of between 5 to 200 mM, 15-200, between 5 to 100 mM,between 15 to 100 mM, between 5 to 50 mM, between 15 to 50 mM, between 5to 25 mM, between 5 to 20 mM, between 5 to 15 mM, or any range thereinbetween. In some embodiments, the buffer is HEPES at a concentration of15 to 200 mM, or any range therein between. In some embodiments, thebuffer is citrate at a concentration of 15 to 200 mM, or any rangetherein between. In some embodiments, the buffer is sodium phosphate ata concentration of 15 to 200 mM, or any range therein between. In someembodiments, the interior space of the liposome comprises a totalconcentration of sodium acetate and calcium acetate of between 5 mM to500 mM, or 50 mM to 500 mM, or any range therein between.

In some embodiments, the interior space of the liposome includestrehalose. In further embodiments, the concentration weight percent oftrehalose is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or 5-20%, or any rangetherein between. In yet further embodiments, the concentration (weightpercent) of trehalose is 1-15%, or any range therein between. In anadditional embodiment, the trehalose is present at about 5% to 20%weight percent of trehalose or any combination of one or morelyoprotectants or cryoprotectants at a total concentration of 5% to 20%.In some embodiments, the pH of solutions comprising the liposomecomposition is from 6 to 7.5, from 6.5 to 7.5, from 6.7 to 7.5, or from6.3 to 7.0, or any range therein between. In some embodiments, theinterior space comprises buffer. In some embodiments, the buffer isselected from HEPES, citrate, or sodium phosphate (e.g., monobasicand/or dibasic sodium phosphate). In some embodiments, the buffer isHEPES. In some embodiments, the buffer is citrate. In some embodiments,the buffer is sodium phosphate (e.g., monobasic and/or dibasic sodiumphosphate). In some embodiments, the buffer is at a concentration of 15to 200 mM, or any range therein between. In yet further embodiments, thebuffer is at a concentration of between 5 to 200 mM, 15-200, between 5to 100 mM, between 15 to 100 mM, between 5 to 50 mM, between 15 to 50mM, between 5 to 25 mM, between 5 to 20 mM, between 5 to 15 mM, or anyrange therein between. In some embodiments, the buffer is HEPES at aconcentration of 15 to 200 mM, or any range therein between. In someembodiments, the buffer is citrate at a concentration of 15 to 200 mM,or any range therein between. In some embodiments, the buffer is sodiumphosphate at a concentration of 15 to 200 mM, or any range thereinbetween. In additional embodiments, the interior space of the liposomecomprises sodium acetate and/or calcium acetate. In some embodiments,the interior space of the liposome comprises a total concentration ofsodium acetate and calcium acetate of between 5 mM to 500 mM, or 50 mMto 500 mM, or any range therein between.

In some embodiments, the interior space of the liposome includesdextrose. In further embodiments, the concentration weight percent ofdextrose is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or 5-20%, or any rangetherein between. In yet further embodiments, the concentration (weightpercent) of dextrose is 1-15%, or any range therein between. In anadditional embodiment, the dextrose is present at about 5% to 20% weightpercent of dextrose or any combination of one or more lyoprotectants orcryoprotectants at a total concentration of 5% to 20%. In someembodiments, the pH of solutions comprising the liposome composition isfrom 6 to 7.5, from 6.5 to 7.5, from 6.7 to 7.5, or from 6.3 to 7.0, orany range therein between. In some embodiments, the interior spacecomprises buffer. In some embodiments, the buffer is selected fromHEPES, citrate, or sodium phosphate (e.g., monobasic and/or dibasicsodium phosphate). In some embodiments, the buffer is HEPES. In someembodiments, the buffer is citrate. In some embodiments, the buffer issodium phosphate (e.g., monobasic and/or dibasic sodium phosphate). Insome embodiments, the buffer is at a concentration of 15 to 200 mM, orany range therein between. In yet further embodiments, the buffer is ata concentration of between 5 to 200 mM, 15-200, between 5 to 100 mM,between 15 to 100 mM, between 5 to 50 mM, between 15 to 50 mM, between 5to 25 mM, between 5 to 20 mM, between 5 to 15 mM, or any range thereinbetween. In some embodiments, the buffer is HEPES at a concentration of15 to 200 mM, or any range therein between. In some embodiments, thebuffer is citrate at a concentration of 15 to 200 mM, or any rangetherein between. In some embodiments, the buffer is sodium phosphate ata concentration of 15 to 200 mM, or any range therein between Inadditional embodiments, the interior space of the liposome comprisessodium acetate and/or calcium acetate. In some embodiments, the interiorspace of the liposome comprises a total concentration of sodium acetateand calcium acetate of between 5 mM to 500 mM, or 50 mM to 500 mM, orany range therein between.

In additional embodiments, the disclosure provides liposomalcompositions that comprise a liposome encapsulating (filled with) aαPTHF (e.g., a αPTHF disclosed herein). In some embodiments, a liposomein the liposomal composition comprises a αPTHF containing 4, 5, 2-10,4-6, or more than 5, glutamyl groups (including the glutamyl group intetrahydrofolate). In additional embodiments, the αPTHF in the Lp-αPTHFcomprises two or more glutamyl groups that have a gamma carboxyllinkage. In some embodiments, the liposomal composition comprises aliposome comprising a tetraglutamated THF. In some embodiments, theliposomal composition comprises a liposome comprising a pentaglutamatedTHF. In some embodiments, the liposomal composition comprises a liposomecomprising a hexaglutamated THF.

In some embodiments, the targeted pegylated liposomal polyglutamatedalpha (e.g., pentaglutamated or hexaglutamated) tetrahydrofolatecomprises a medium comprising a liposome including an interior space; anaqueous αPTHF disposed within the interior space; and a targeting moietycomprising a protein with specific affinity for at least one folatereceptor, and wherein the targeting moiety disposed at the exterior ofthe liposome. In some embodiments, the medium is an aqueous solution. Insome embodiments, the interior space, the exterior space (e.g., themedium), or both the interior space and the medium contains one or morelyoprotectants or cryoprotectants which are listed above. In someembodiments, the cryoprotectant is mannitol, trehalose, sorbitol, orsucrose.

In some embodiments, the liposome encapsulating αPTHF (i.e., Lp-αPTHF,including PLp-αPTHF, TPLp-αPTHF, TLp-αPTHF, and NTLp-αPTHF) has aninterior space that contains less than 500,000 or less than 200,000molecules of αPTHF. In some embodiments, the liposome interior spacecontains between 10 to 100,000 molecules of αPTHF, or any range thereinbetween. In some embodiments, the liposome interior space containsbetween 10,000 to 100,000 molecules of αPTHF, or any range thereinbetween. In some embodiments, the liposome is unpegylated and has aninterior space that contains less than 500,000 or less than 200,000molecules of αPTHF. In some embodiments, the liposome is unpegylated andthe interior space of the liposome contains between 10 to 100,000molecules of αPTHF, or any range therein between. In furtherembodiments, the liposome is unpegylated and the interior space of theliposome contains between 10,000 to 100,000 molecules of αPTHF, or anyrange therein between. In some embodiments, the liposome is targeted andunpegylated (TLp-αPTHF) and has an interior space that contains lessthan 500,000 or less than 200,000 molecules of αPTHF. In someembodiments, the liposome is targeted and unpegylated and the interiorspace of the liposome contains between 10 to 100,000 molecules of αPTHF,or any range therein between. In further embodiments, the liposome istargeted and unpegylated and the interior space of the liposome containsbetween 10,000 to 100,000 molecules of αPTHF, or any range thereinbetween. In some embodiments, the liposome is non-targeted andunpegylated (NTLp-αPTHF) and has an interior space that contains lessthan 500,000 or less than 200,000 molecules of αPTHF. In someembodiments, the liposome is non-targeted and unpegylated and theinterior space of the liposome contains between 10 to 100,000 moleculesof αPTHF, or any range therein between. In further embodiments, theliposome is non-targeted and unpegylated and the interior space of theliposome contains between 10,000 to 100,000 molecules of αPTHF, or anyrange therein between.

In some embodiments, the liposome encapsulates polyglutamated alphacontaining 2-10 glutamyl groups (i.e., Lp-αPTHF, including PLp-αPTHF,TPLp-αPTHF, TLp-αPTHF, and NTLp-αPTHF) and has an interior space thatcontains less than 500,000 or 200,000 molecules of αPTHF containing 2-10glutamyl groups. In some embodiments, the liposome interior spacecontains between 10 to 100,000 molecules of αPTHF containing 2-10glutamyl groups, or any range therein between. In further embodiments,the liposome interior space contains between 10,000 to 100,000 moleculesof αPTHF containing 2-10 glutamyl groups, or any range therein between.In some embodiments, the liposome is unpegylated and has an interiorspace that contains less than 500,000 or 200,000 molecules of αPTHFcontaining 2-10 glutamyl groups. In some embodiments, the liposome isunpegylated and the interior space of the liposome contains between 10to 100,000 molecules of αPTHF containing 2-10 glutamyl groups, or anyrange therein between. In further embodiments, the liposome isunpegylated and the interior space of the liposome contains between10,000 to 100,000 molecules of αPTHF containing 2-10 glutamyl groups, orany range therein between. In some embodiments, the liposome is targetedand unpegylated (TLp-αPTHF) and has an interior space that contains lessthan 500,000 or 200,000 molecules of αPTHF containing 2-10 glutamylgroups. In some embodiments, the liposome is targeted and unpegylatedand the interior space of the liposome contains between 10 to 100,000molecules αPTHF containing 2-10 glutamyl groups, or any range thereinbetween. In further embodiments, the liposome is targeted andunpegylated and the interior space of the liposome contains between10,000 to 100,000 molecules αPTHF containing 2-10 glutamyl groups, orany range therein between. In some embodiments, the liposome isnon-targeted and unpegylated (NTLp-αPTHF) and has an interior space thatcontains less than 500,000 or 200,000 molecules of αPTHF containing 2-10glutamyl groups. In some embodiments, the liposome is non-targeted andunpegylated and the interior space of the liposome contains between 10to 100,000 molecules of αPTHF containing 2-10 glutamyl groups, or anyrange therein between. In further embodiments, the liposome isnon-targeted and unpegylated and the interior space of the liposomecontains between 10,000 to 100,000 molecules of αPTHF containing 2-10glutamyl groups, or any range therein between.

In some embodiments, the liposome encapsulates tetraglutamated alphatetrahydrofolate (i.e., Lp-αPTHF, including PLp-αPTHF, TPLp-αPTHF,TLp-αPTHF, and NTLp-αPTHF) and has an interior space that contains lessthan 500,000 or 200,000 molecules of tetraglutamated alphatetrahydrofolate. In some embodiments, the liposome interior spacecontains between 10 to 100,000 molecules of tetraglutamated alphatetrahydrofolate, or any range therein between. In some embodiments, theliposome interior space contains between 10,000 to 100,000 molecules oftetraglutamated alpha tetrahydrofolate, or any range therein between. Insome embodiments, the liposome is unpegylated and has an interior spacethat contains less than 500,000 or 200,000 molecules of tetraglutamatedalpha tetrahydrofolate. In some embodiments, the liposome is unpegylatedand the interior space of the liposome contains between 10 to 100,000molecules of tetraglutamated alpha tetrahydrofolate, or any rangetherein between. In further embodiments, the liposome is unpegylated andthe interior space of the liposome contains between 10,000 to 100,000molecules of tetraglutamated alpha tetrahydrofolate, or any rangetherein between. In some embodiments, the liposome is targeted andunpegylated (TLp-αPTHF) and has an interior space that contains lessthan 500,000 or 200,000 molecules of tetraglutamated alphatetrahydrofolate. In some embodiments, the liposome is targeted andunpegylated and the interior space of the liposome contains between 10to 100,000 molecules of tetraglutamated alpha tetrahydrofolate, or anyrange therein between. In further embodiments, the liposome is targetedand unpegylated and the interior space of the liposome contains between10,000 to 100,000 molecules of tetraglutamated alpha tetrahydrofolate,or any range therein between. In some embodiments, the liposome isnon-targeted and unpegylated (NTLp-αPTHF) and has an interior space thatcontains less than 500,000 or 200,000 molecules of tetraglutamated alphatetrahydrofolate. In some embodiments, the liposome is non-targeted andunpegylated and the interior space of the liposome contains between 10to 100,000 molecules of tetraglutamated alpha tetrahydrofolate, or anyrange therein between. In further embodiments, the liposome isnon-targeted and unpegylated and the interior space of the liposomecontains between 10,000 to 100,000 molecules of tetraglutamated alphatetrahydrofolate, or any range therein between.

In some embodiments, the liposome encapsulates pentaglutamated alphatetrahydrofolate (i.e., Lp-αPTHF, including PLp-αPTHF, TPLp-αPTHF,TLp-αPTHF, and NTLp-αPTHF) and has an interior space that contains lessthan 500,000 or 200,000 molecules of pentaglutamated alphatetrahydrofolate. In some embodiments, the liposome interior spacecontains between 10 to 100,000 molecules of pentaglutamated alphatetrahydrofolate, or any range therein between. In some embodiments, theliposome interior space contains between 10,000 to 100,000 molecules ofpentaglutamated alpha tetrahydrofolate, or any range therein between. Insome embodiments, the liposome is unpegylated and has an interior spacethat contains less than 500,000 or 200,000 molecules of pentaglutamatedalpha tetrahydrofolate. In some embodiments, the liposome is unpegylatedand the interior space of the liposome contains between 10 to 100,000molecules of pentaglutamated alpha tetrahydrofolate, or any rangetherein between. In further embodiments, the liposome is unpegylated andthe interior space of the liposome contains between 10,000 to 100,000molecules of pentaglutamated alpha tetrahydrofolate, or any rangetherein between. In some embodiments, the liposome is targeted andunpegylated (TLp-αPTHF) and has an interior space that contains lessthan 500,000 or 200,000 molecules of pentaglutamated alphatetrahydrofolate. In some embodiments, the liposome is targeted andunpegylated and the interior space of the liposome contains between 10to 100,000 molecules of pentaglutamated alpha tetrahydrofolate, or anyrange therein between. In further embodiments, the liposome is targetedand unpegylated and the interior space of the liposome contains between10,000 to 100,000 molecules of pentaglutamated alpha tetrahydrofolate,or any range therein between. In some embodiments, the liposome isnon-targeted and unpegylated (NTLp-αPTHF) and has an interior space thatcontains less than 500,000 or 200,000 molecules of pentaglutamated alphatetrahydrofolate. In some embodiments, the liposome is non-targeted andunpegylated and the interior space of the liposome contains between 10to 100,000 molecules of pentaglutamated alpha tetrahydrofolate, or anyrange therein between. In further embodiments, the liposome isnon-targeted and unpegylated and the interior space of the liposomecontains between 10,000 to 100,000 molecules of pentaglutamated alphatetrahydrofolate, or any range therein between.

In some embodiments, the liposome encapsulates hexaglutamated alphatetrahydrofolate (i.e., Lp-αPTHF, including PLp-αPTHF, TPLp-αPTHF,TLp-αPTHF, and NTLp-αPTHF) and has an interior space that contains lessthan 500,000 or 200,000 molecules of hexaglutamated alphatetrahydrofolate. In some embodiments, the liposome interior spacecontains between 10 to 100,000 molecules of hexaglutamated alphatetrahydrofolate, or any range therein between. In further embodiments,the liposome interior space contains between 10,000 to 100,000 moleculesof hexaglutamated alpha tetrahydrofolate, or any range therein between.In some embodiments, the liposome is unpegylated and has an interiorspace that contains less than 500,000 or 200,000 molecules ofhexaglutamated alpha tetrahydrofolate. In some embodiments, the liposomeis unpegylated and the interior space of the liposome contains between10 to 100,000 molecules of hexaglutamated alpha tetrahydrofolate, or anyrange therein between. In further embodiments, the liposome isunpegylated and the interior space of the liposome contains between10,000 to 100,000 molecules of hexaglutamated alpha tetrahydrofolate, orany range therein between. In some embodiments, the liposome is targetedand unpegylated (TLp-αPTHF) and has an interior space that contains lessthan 500,000 or 200,000 molecules of hexaglutamated alphatetrahydrofolate. In some embodiments, the liposome is targeted andunpegylated and the interior space of the liposome contains between 10to 100,000 molecules of hexaglutamated alpha tetrahydrofolate, or anyrange therein between. In further embodiments, the liposome is targetedand unpegylated and the interior space of the liposome contains between10,000 to 100,000 molecules of hexaglutamated alpha tetrahydrofolate, orany range therein between. In some embodiments, the liposome isnon-targeted and unpegylated (NTLp-αPTHF) and has an interior space thatcontains less than 500,000 or 200,000 molecules of hexaglutamated alphatetrahydrofolate. In some embodiments, the liposome is non-targeted andunpegylated and the interior space of the liposome contains between 10to 100,000 molecules of hexaglutamated alpha tetrahydrofolate, or anyrange therein between. In further embodiments, the liposome isnon-targeted and unpegylated and the interior space of the liposomecontains between 10,000 to 100,000 molecules of hexaglutamated alphatetrahydrofolate, or any range therein between.

In some embodiments, the disclosure provides a liposomal αPTHFcomposition wherein the liposome encapsulates αPTHF or a salt or acidthereof, and one or more aqueous pharmaceutically acceptable carriers.In some embodiments, the liposome interior space contains trehalose. Insome embodiments, the liposome interior space contains 5% to 20% weightof trehalose. In some embodiments, the liposome interior space containsHBS at a concentration of between 1 to 200 mM and a pH of between 2 to8. In some embodiments, liposome interior space has a pH 5-8, or anyrange therein between. In some embodiments, liposome interior space hasa pH 6-7, or any range therein between. In some embodiments, theliposome interior space has a total concentration of sodium acetate andcalcium acetate of between 50 mM to 500 mM, or any range thereinbetween.

A Non-Polyglutamated Polyglutamatable Antifolates

In some embodiments, the liposome αPTHF (e.g., Lp-αPTHF, includingPLp-αPTHF, TPLp-αPTHF, TLp-αPTHF, and NTLp-αPTHF) compositions compriseαPTHF e.g., a αPTHF disclosed herein) and one or morenon-polyglutamated, polyglutamatable antifolate compositions.

In some embodiments, the Lp-αPTHF (e.g., PLp-αPTHF, TPLp-αPTHF,TLp-αPTHF, and NTLp-αPTHF) comprises αPTHF (e.g., a αPTHF disclosedherein) and tetrahydrofolate (THF). In some embodiments, the Lp-αPTHF(i.e., liposome alpha polyglutamated tetrahydrofolate) comprises alphapolyglutamated tetrahydrofolate and a polyglutamatable antifolateselected from the group consisting of: tetrahydrofolate (THF),methotrexate (MTX), pemetrexed (PMX), lometrexol (LMX), raltitrexed(RTX), pralatrexate, AG2034, GW1843, aminopterin, and LY309887. In someembodiments, the Lp-αPTHF comprises alpha polyglutamatedtetrahydrofolate and lometrexol. In some embodiments, the Lp-αPTHFcomprises alpha polyglutamated tetrahydrofolate and pemetrexed. In someembodiments, the Lp-αPTHF comprises alpha polyglutamatedtetrahydrofolate and leucovorin. In some embodiments, the Lp-αPTHFcomprises alpha polyglutamated tetrahydrofolate and a triazineantifolate derivative (e.g., a sulphonyl fluoride triazine such as NSC127755). In some embodiments, the Lp-αPTHF comprises alphapolyglutamated tetrahydrofolate and a serine hydroxymethyltransferase(SHMT2) inhibitor. In some embodiments, the SHMT2 inhibitor is anantifolate (e.g., a polyglutamatable or nonpolyglutamatable antifolate).In some embodiments, the SHMT2 inhibitor is an antifolate.

B Non-Polyglutamatable Antifolates

In some embodiments, the Lp-αPTHF (e.g., PLp-αPTHF, TPLp-αPTHF,TLp-αPTHF, and NTLp-αPTHF) comprises a αPTHF (e.g., a αPTHF disclosedherein) and a so-called “non-polyglutamatable” antifolate. In someembodiments, the liposome comprises a αPTHF and a non-polyglutamatableantifolate that inhibits one or more enzymes in the folate cyclemetabolic pathway. In further embodiments, the non-polyglutamatableantifolate inhibits one or more enzymes selected from: thymidylatesynthase (TS), dihydrofolate reductase (DHFR), glycinamideribonucleotide (GAR) transformylase, and aminoimidazole carboxamideribonucleotide (AICAR) transformylase. In some embodiments, the liposomecomprises a αPTHF and a non-polyglutamatable antifolate that inhibitsDHFR. In some embodiments, the liposome comprises a αPTHF and anon-polyglutamatable antifolate that inhibits TS. In some embodiments,the liposome comprises a αPTHF and a non-polyglutamatable antifolatethat inhibits GAR or AICAR transformylase. In further embodiments, thenon-polyglutamatable antifolate is selected from the group consistingof: trimetrexate (TMQ), piritrexim (BW301U), and talotrexin (PT523). Infurther embodiments, the non-polyglutamatable antifolate is selectedfrom the group consisting of: nolatrexed (AG337), plevitrexed (ZD9331,BGC9331), and BGC 945 (ONX 0801).

C Platinums

In some embodiments, the liposome comprises a αPTHF (Lp-αPTHF, such ase.g., PLp-αPTHF, TPLp-αPTHF, TLp-αPTHF, and NTLp-αPTHF) comprises aαPTHF (e.g., a αPTHF disclosed herein) and a platinum-basedchemotherapeutic agent or a salt or acid, thereof. In some embodiments,the liposome contains a αPTHF/platinum based agent complex (e.g., asdescribed in Section IIC).

In some embodiments, the Lp-αPTHF comprises a platinum-basedchemotherapeutic agent selected from the group consisting of: cisplatin,carboplatin, and oxaliplatin, or a salt or acid thereof. In otherembodiments, the Lp-αPTHF comprises an analog of a platinum-basedchemotherapeutic agent selected from the group consisting of: cisplatin,carboplatin, or oxaliplatin, or a salt or acid thereof.

In some embodiments, the Lp-αPTHF comprises a αPTHF and cisplatin or asalt or acid thereof. In some embodiments, the Lp-αPTHF comprises aαPTHF and a cisplatin analog, or a salt or acid thereof.

In some embodiments, the Lp-αPTHF comprises a αPTHF and carboplatin, ora salt or acid thereof. In some embodiments, the liposome comprises aαPTHF and carboplatin analog, or a salt or acid thereof.

In some embodiments, the Lp-αPTHF comprises a αPTHF and oxaliplatin, ora salt or acid thereof. In some embodiments, the liposome comprises aαPTHF and an oxaliplatin analog, or a salt or acid thereof.

In some embodiments, the liposome comprises a αPTHF (e.g., a αPTHFdisclosed herein) and a platinum-based chemotherapeutic agent selectedfrom the group consisting of: nedaplatin, heptaplatin, and lobaplatin,nedaplatin, heptaplatin, and lobaplatin or a salt or acid thereof. Insome embodiments, the Lp-αPTHF comprises a αPTHF and an analog of aplatinum-based chemotherapeutic agent selected from the group consistingof: nedaplatin, heptaplatin, and lobaplatin, or a salt or acid thereof.

In some embodiments, the Lp-αPTHF comprises a αPTHF and a platinum-basedchemotherapeutic agent selected from the group consisting of:stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin,spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin,zeniplatin, platinum-triamine, traplatin, enloplatin, JM216, 254S,NK121, CI973, DWA 2114R, NDDP, and dedaplatin, or a salt or acidthereof. In some embodiments, the Lp-αPTHF comprises a αPTHF and ananalog of a platinum-based chemotherapeutic agent selected from thegroup consisting of: stratoplatin, paraplatin, platinol, cycloplatin,dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin,iproplatin, ormaplatin, zeniplatin, platinum-triamine, traplatin,enloplatin, JM216, 254S, NK121, CI973, DWA 2114R, NDDP, and dedaplatin,or a salt or acid thereof.

In some embodiments, the liposome composition comprises liposomes thatfurther contain one or more of an immunostimulatory agent, a detectablemarker and a maleimide disposed on at least one of the PEG and theexterior of the liposome.

D Cyclodextrins

In additional embodiments, the αPTHF liposome comprise a αPTHF (e.g., aαPTHF disclosed herein) and a cyclodextrin (e.g., a cyclodextrin inSection IIC, herein).

In some embodiments, the αPTHF liposome comprises a complex formed by acyclodextrin and a therapeutic agent. In some embodiments, thetherapeutic agent is a cytotoxic compound or a salt or acid thereof. Ina further embodiment, the therapeutic agent is a chemotherapeutic agentor a salt or acid thereof. In another embodiment, the therapeutic agentis a platinum-based drug. In another embodiment, the therapeutic agentis a taxane-based drug. In further embodiments, the therapeutic agent ofthe cyclodextrin/therapeutic agent complex is a member selected from thegroup consisting of: gemcitabine, a gemcitabine-based therapeutic agent,doxorubicin, an antifolate, an antifolate-based chemotherapeutic, or asalt or acid, acid or free base form thereof. In additional embodiments,the molar ratio of cyclodextrin/therapeutic agent in the complex is inthe range 1-10:1. In some embodiments, the molar ratio ofcyclodextrin/therapeutic agent in the complex is 1:1, 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molarratio of cyclodextrin/therapeutic agent in the complex is in the range1:1-20, 1:1-10, or 1:2-8, or any range therein between. In someembodiments, the molar ratio of cyclodextrin/therapeutic agent is: 1:1,1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14,1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50.

In some embodiments, the αPTHF liposome comprises αPTHF and acyclodextrin/platinum-based chemotherapeutic agent complex. In someembodiments, the platinum-based chemotherapeutic agent is selected fromthe group consisting of: cisplatin, carboplatin, and oxaliplatin, or asalt or acid thereof. In other embodiments, thecyclodextrin/platinum-based chemotherapeutic agent complex comprises ananalog of a cisplatin, carboplatin, oxaliplatin, or a salt or acidthereof. In some embodiments, the molar ratio ofcyclodextrin/platinum-based agent in the complex is in the range 1-10:1.In some embodiments, the molar ratio of cyclodextrin/platinum-basedagent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In other embodiments, the molar ratio ofcyclodextrin/platinum-based agent in the complex is in the range 1:1-20,1:1-10, or 1:2-8, or any range therein between. In some embodiments, themolar ratio of cyclodextrin/platinum-based agent is: 1:1, 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:(21-50), or 1:>50.

In some embodiments, the platinum-based chemotherapeutic agent isselected from the group consisting of: cisplatin, carboplatin, andoxaliplatin, or a salt or acid thereof. In other embodiments, thecyclodextrin/platinum-based chemotherapeutic agent complex comprises ananalog of a cisplatin, carboplatin, oxaliplatin, or a salt or acidthereof. In some embodiments, the molar ratio ofcyclodextrin/platinum-based agent in the complex is in the range 1-10:1.In some embodiments, the molar ratio of cyclodextrin/platinum-basedagent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In other embodiments, the molar ratio ofcyclodextrin/platinum-based agent in the complex is in the range 1:1-20,1:1-10, or 1:2-8, or any range therein between. In some embodiments, themolar ratio of cyclodextrin/platinum-based agent is: 1:1, 1:2, 1:3, 1:4,1:5,1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments, thecyclodextrin/platinum-based agent complex is encapsulated in a liposome(e.g., as described herein or otherwise known in the art).

In further embodiments, the disclosure provides a complex containingcyclodextrin and cisplatin or a salt or acid thereof. In someembodiments, the molar ratio of cyclodextrin/cisplatin (or cisplatinsalt or acid) in the complex is in the range 1-10:1. In someembodiments, the molar ratio of cyclodextrin/cisplatin (or cisplatinsalt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In other embodiments, the molar ratio ofcyclodextrin/cisplatin (or cisplatin salt or acid) in the complex is inthe range 1:1-20, 1:1-10, or 1:2-8, or any range therein between. Insome embodiments, the molar ratio of cyclodextrin/cisplatin (orcisplatin salt or acid) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:(21-50), or 1:>50. In additional embodiments, thecyclodextrin/cisplatin (or cisplatin salt or acid) complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In another embodiment, the disclosure provides a complex containingcyclodextrin and carboplatin or a salt or acid thereof. In someembodiments, the molar ratio of cyclodextrin/carboplatin (or carboplatinsalt or acid) in the complex is in the range 1-10:1. In someembodiments, the molar ratio of cyclodextrin/carboplatin (or carboplatinsalt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In other embodiments, the molar ratio ofcyclodextrin/carboplatin (or carboplatin salt or acid) in the complex isin the range 1:1-20, 1:1-10, or 1:2-8, or any range therein between. Insome embodiments, the molar ratio of cyclodextrin/carboplatin (orcarboplatin salt or acid) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:(21-50), or 1:>50. In additional embodiments, thecyclodextrin/carboplatin (or carboplatin salt or acid) complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In another embodiment, the disclosure provides a complex containingcyclodextrin and oxaliplatin, or a salt or acid thereof. In someembodiments, the molar ratio of cyclodextrin/oxaliplatin (or oxaliplatinsalt or acid) in the complex is in the range 1-10:1. In someembodiments, the molar ratio of cyclodextrin/oxaliplatin (or oxaliplatinsalt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In other embodiments, the molar ratio ofcyclodextrin/oxaliplatin (or oxaliplatin salt or acid) in the complex isin the range 1:1-20, 1:1-10, or 1:2-8, or any range therein between. Insome embodiments, the molar ratio of cyclodextrin/oxaliplatin (oroxaliplatin salt or acid) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:(21-50), or 1:>50. In additional embodiments, thecyclodextrin/oxaliplatin (or oxaliplatin salt or acid) complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In additional embodiments, the disclosure provides a complex comprisingcyclodextrin and a platinum-based chemotherapeutic agent selected fromthe group consisting of: nedaplatin, heptaplatin, lobaplatin,stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin,spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin,zeniplatin, platinum-triamine, traplatin, enloplatin, JM216, NK121,CI973, DWA 2114R, NDDP, and dedaplatin, or a salt or acid thereof. Inother embodiments, the cyclodextrin/platinum-based chemotherapeuticagent complex comprises an analog of nedaplatin, heptaplatin,lobaplatin, stratoplatin, paraplatin, platinol, cycloplatin,dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin,iproplatin, ormaplatin, zeniplatin, platinum-triamine, traplatin,enloplatin, JM216, NK121, CI973, DWA 2114R, NDDP, or dedaplatin, or asalt or acid thereof. In some embodiments, the molar ratio ofcyclodextrin/oxaliplatin (or oxaliplatin salt or acid) in the complex isin the range 1-10:1. In some embodiments, the molar ratio ofcyclodextrin/platinum-based chemotherapeutic agent (or salt or acid oranalog thereof) in the complex 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In other embodiments, the molar ratio ofcyclodextrin/platinum-based chemotherapeutic agent (or salt or acid oranalog thereof) in the complex is in the range 1:1-20, 1:1-10, or 1:2-8,or any range therein between. In some embodiments, the molar ratio ofcyclodextrin/platinum-based chemotherapeutic agent (or salt or acid oranalog thereof) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10,1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50),or 1:>50. In additional embodiments, the cyclodextrin/platinum-basedchemotherapeutic agent (or salt or acid or analog thereof) complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In some embodiments, the disclosure provides a composition comprising acyclodextrin/taxane-based chemotherapeutic agent complex. In someembodiments, the taxane-based chemotherapeutic agent is selected fromthe group consisting of: paclitaxel (PTX), docetaxel (DTX), larotaxel(LTX), and cabazitaxel (CTX), or a salt or acid thereof. In someembodiments, the molar ratio of cyclodextrin/taxane-based agent in thecomplex is in the range 1-10:1. In some embodiments, the molar ratio ofcyclodextrin/taxane-based agent in the complex is 1:1, 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molarratio of cyclodextrin/taxane-based agent in the complex is in the range1:1-20, 1:1-10, or 1:2-8, or any range therein between. In someembodiments, the molar ratio of cyclodextrin/taxane-based agent is: 1:1,1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14,1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additionalembodiments, the cyclodextrin/taxane-based agent complex is encapsulatedin a liposome (e.g., as described herein or otherwise known in the art).

In additional embodiments, the disclosure provides a complex comprisingcyclodextrin and paclitaxel (PTX), or a salt or acid thereof. In otherembodiments, the cyclodextrin/taxane-based chemotherapeutic agentcomplex comprises an analog of paclitaxel (PTX), or a salt or acidthereof. In some embodiments, the molar ratio of cyclodextrin/paclitaxel(or paclitaxel salt or acid) in the complex is in the range 1-10:1. Insome embodiments, the molar ratio of cyclodextrin/paclitaxel (orpaclitaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1,19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratioof cyclodextrin/paclitaxel (or paclitaxel salt or acid) in the complexis in the range 1:1-20, 1:1-10, or 1:2-8, or any range therein between.In some embodiments, the molar ratio of cyclodextrin/paclitaxel (orpaclitaxel salt or acid) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:(21-50), or 1:>50. In additional embodiments, thecyclodextrin/paclitaxel (or paclitaxel salt or acid) complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In additional embodiments, the disclosure provides a complex comprisingcyclodextrin and docetaxel (DTX), or a salt or acid thereof. In otherembodiments, the cyclodextrin/taxane-based chemotherapeutic agentcomplex comprises an analog of docetaxel (DTX), or a salt or acidthereof. In some embodiments, the molar ratio of cyclodextrin/docetaxel(or docetaxel salt or acid) in the complex is in the range 1-10:1. Insome embodiments, the molar ratio of cyclodextrin/docetaxel (ordocetaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1,19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratioof cyclodextrin/docetaxel (or docetaxel salt or acid) in the complex isin the range 1:1-20, 1:1-10, or 1:2-8, or any range therein between. Insome embodiments, the molar ratio of cyclodextrin/docetaxel (ordocetaxel salt or acid) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:(21-50), or 1:>50. In additional embodiments, thecyclodextrin/docetaxel (or docetaxel salt or acid) complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In additional embodiments, the disclosure provides a complex comprisingcyclodextrin and larotaxel (LTX), or a salt or acid thereof. In otherembodiments, the cyclodextrin/taxane-based chemotherapeutic agentcomplex comprises an analog of larotaxel (LTX), or a salt or acidthereof. In some embodiments, the molar ratio of cyclodextrin/larotaxel(or larotaxel salt or acid) in the complex is in the range 1-10:1. Insome embodiments, the molar ratio of cyclodextrin/larotaxel (orlarotaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1,19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratioof cyclodextrin/larotaxel (or larotaxel salt or acid) in the complex isin the range 1:1-20, 1:1-10, or 1:2-8, or any range therein between. Insome embodiments, the molar ratio of cyclodextrin/larotaxel (orlarotaxel salt or acid) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:(21-50), or 1:>50. In additional embodiments, thecyclodextrin/larotaxel (or larotaxel salt or acid) complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In additional embodiments, the disclosure provides a complex comprisingcyclodextrin and cabazitaxel (CTX), or a salt or acid thereof. In otherembodiments, the cyclodextrin/taxane-based chemotherapeutic agentcomplex comprises an analog of cabazitaxel (CTX), or a salt or acidthereof. In some embodiments, the molar ratio ofcyclodextrin/cabazitaxel (or cabazitaxel salt or acid) in the complex isin the range 1-10:1. In some embodiments, the molar ratio ofcyclodextrin/cabazitaxel (or cabazitaxel salt or acid) in the complex is1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1,14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In otherembodiments, the molar ratio of cyclodextrin/cabazitaxel (or cabazitaxelsalt or acid) in the complex is in the range 1:1-20, 1:1-10, or 1:2-8,or any range therein between. In some embodiments, the molar ratio ofcyclodextrin/cabazitaxel (or cabazitaxel salt or acid) is: 1:1, 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15,1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additionalembodiments, the cyclodextrin/cabazitaxel (or cabazitaxel salt or acid)complex is encapsulated in a liposome (e.g., as described herein orotherwise known in the art).

The cyclodextrin of the cyclodextrin/therapeutic agent complex can bederivatized or underivatized. In some embodiments, the cyclodextrin isderivatized. In further embodiments, the cyclodextrin is a derivatizedbeta-cyclodextrin (e.g., a hydroxypropyl beta-cyclodextrin (HP-beta-CD),and a sulfobutyl ether beta-CD (SBE)-beta-cyclodextrin). In someembodiments, the cyclodextrin of the cyclodextrin/therapeutic agentcomplex is a derivatized beta-cyclodextrin comprising: 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more 2-hydroxylpropyl-3-group substitutions of hydroxygroups; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sulfoalkyl ether groupsubstitutions of hydroxy groups. In further embodiments, thecyclodextrin of the cyclodextrin/therapeutic agent complex is aderivatized beta-cyclodextrin comprising: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more sulfobutyl ether group substitutions of hydroxy groups.

In some embodiments, the cyclodextrin of the cyclodextrin/therapeuticagent complex contained in the αPTHF liposome composition is aderivatized cyclodextrin of Formula I:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —H, a straight chain or branchedC₁-C₈-alkylene group, a 2-hydroxylpropyl-3- group; or an optionallysubstituted straight-chain or branched C₁-C₆ group, wherein at least oneof R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branchedC₁-C₈-alkylene group or a 2-hydroxylpropyl-3- group.

In some embodiments, the cyclodextrin of the cyclodextrin/therapeuticagent complex contained in the αPTHF liposome composition is aderivatized cyclodextrin of Formula II:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —O— or a —O—(C2-C6 alkylene)-SO3⁻ group;wherein at least one of R₁ and R₂ is independently a —O—(C₂-C₆alkylene)-SO3⁻ group; and S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ areeach, independently, a —H or a H or a pharmaceutically acceptablecation. In further embodiments, the wherein the pharmaceuticallyacceptable cation is selected from: an alkali metal such as Li⁺, Na⁺, orK⁺; an alkaline earth metal such as Ca⁺², or Mg⁺², and ammonium ions andamine cations such as the cations of (C1-C6)-alkylamines, piperidine,pyrazine, (C1-C6)-alkanolamine and (C4-C8)-cycloalkanolamine.

In some embodiments, the αPTHF liposome comprises between 100 to 100,000of the cyclodextrin/therapeutic agent complexes.

In some embodiments, a cyclodextrin derivative of the αPTHF/cyclodextrincomplex and/or cyclodextrin/therapeutic agent complex is a cyclodextrindisclosed in U.S. Pat. Nos. 6,133,248, 5,874,418, 6,046,177, 5,376,645,5,134,127, 7,034,013, 6,869,939; and Intl. Appl. Publ. No. WO02005/117911, the contents each of which is herein incorporated byreference in its priority.

In some embodiments, the cyclodextrin derivative of thecyclodextrin/therapeutic agent complex is a sulfoalkyl ethercyclodextrin. In some embodiments, the cyclodextrin derivative ofcomplex is a sulfobutyl ether-3-cyclodextrin such as CAPTISOL® (CyDexPharma. Inc., Lenexa, Kans. Methods for preparing sulfobutylether-3-cyclodextrin and other sulfoalkyl ether cyclodextrins are knownin the art.

In some embodiments, the cyclodextrin derivative of thecyclodextrin/therapeutic agent complex is a compound of Formula III:

wherein R equals:

(e) (H)_(21-X) or (—(CH₂)₄—SO₃Na)x, and x=1.0-10.0, 1.0-5.0, 6.0-7.0, or8.0-10.0;

(f) (H)_(21-X) or (—(CH₂CH(OH)CH₃)x, and x=1.0-10.0, 1.0-5.0, 6.0-7.0,or 8.0-10.0;

(g) (H)_(21-X) or (sulfoalkyl ethers)x, and x=1.0-10.0, 1.0-5.0,6.0-7.0, or 8.0-10.0; or

(h) (H)_(21-X) or (—(CH₂)₄—SO₃Na)x, and x=1.0-10.0, 1.0-5.0, 6.0-7.0, or8.0-10.0.

Additional cyclodextrins and cyclodextrin/platinum-based therapeuticcomplexes that can be contained in the αPTHF liposomes and usedaccording to the disclosed methods is disclosed in U.S. Appl. No.62/583,432, the contents of which is herein incorporated by reference itits entirety.

In some embodiments, the αPTHF liposome comprises a complex of acyclodextrin and a platinum-based chemotherapeutic agent, or a saltthereof. In some embodiments, the platinum-based chemotherapeutic agentis cisplatin or a cisplatin analog. In some embodiments, theplatinum-based chemotherapeutic agent is carboplatin. In additionalembodiments, the liposome composition comprises a platinum-basedchemotherapeutic agent is a member selected from the group consistingof: carboplatin, cisplatin, oxaliplatin, satraplatin, picoplatin,nedaplatin, triplatin, tetraplatin, lipoplatin, lobaplatin, ormaplatin,zeniplatin, platinum-triamine, traplatin, enloplatin, JM216, 254S,NK121, CI973, DWA 2114R, NDDP, and dedaplatin. In some embodiments, theαPTHF liposome comprises between 100 to 100,000 platinum-basedchemotherapeutic agent/CD complexes. In additional embodiments, theliposome composition comprises liposomes that have a diameter in therange of 20 nm to 500 nm or 20 nm to 200 nm, or any range thereinbetween. In some embodiments, liposomes in the composition comprisebetween 100 to 100,000 platinum.

(3) Targeted Liposomes

In some embodiments, the disclosure provides a liposomal αPTHFcomposition wherein the liposome comprises a αPTHF and a targetingmoiety attached to one or both of a PEG and the exterior of theliposome, and wherein the targeting moiety has a specific affinity for asurface antigen on a target cell of interest. Such liposomes maygenerally be referred to herein as “targeted liposomes”, e.g., liposomesincluding one or more targeting moieties or biodistribution modifiers onthe surface of, or otherwise attached to, the liposomes. The targetingmoiety of the targeted liposomes can be any moiety or agent that iscapable of specifically binding a desired target (e.g., an antigentarget expressed on the surface of a target cell of interest). In oneembodiment, the targeted liposome specifically and preferentially bindsto a target on the surface of a target cell of interest thatinternalizes the targeted liposome into which the liposome encapsulatedαPTHF (e.g., pentaglutamated alpha THF or hexaglutamated alpha THF)exerts its cytotoxic effect. In further embodiments, the target cell isa cancer cell, a tumor cell or a metastatic cell. In some embodiments,the targeted liposome is pegylated.

The term “attach” or “attached” refers, for example, to any type ofbonding such as covalent bonding, ionic bonding (e.g., avidin-biotin)bonding by hydrophobic interactions, and bonding via functional groupssuch as maleimide, or linkers such as PEG. For example, a detectablemarker, a steric stabilizer, a liposome, a liposomal component, animmunostimulating agent may be attached to each other directly, by amaleimide functional group, or by a PEG-malemide group.

The composition and origination of the targeting moiety is non-limitingto the scope of this disclosure. In some embodiments, the targetingmoiety attached to the liposome is a polypeptide or peptidomimeticligand. Peptide and peptidomimetic targeting moieties include thosehaving naturally occurring or modified peptides, e.g., D or L peptides;alpha, beta, or gamma peptides; N-methyl peptides; azapeptides; peptideshaving one or more amide, i.e., peptide, linkages replaced with one ormore urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclicpeptides. A peptidomimetic is a molecule capable of folding into adefined three-dimensional structure similar to a natural peptide. Insome embodiments, the peptide or peptidomimetic targeting moiety is 2-50amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50amino acids long

In some embodiments, the targeting moiety polypeptide is at least 40amino acid residues in length. In other embodiments, the targetingmoiety polypeptide is at least 50, 60, 75, 100, 125, 150, 175, 200, 250,or 300 amino acid residues in length.

In additional embodiments, the targeting moiety polypeptide such as anantibody or an antigen-binding antibody fragment that binds a targetantigen with an equilibrium dissociation constant (Kd) in a range of0.5×10⁻¹⁰ to 10×10⁻⁶ as determined using BIACORE® analysis.

In some embodiments, the targeting moiety is an antibody or an antibodyderivative. In other embodiments, the binding domain of the targetingmoiety polypeptide is not derived from the antigen binding domain of anantibody. In some embodiments, the targeting moiety is a polypeptidederived from a binding scaffold selected from the group consisting of aDARPin, affilin, and armadillo repeat, D domain (see, e.g., WO2016/164308), Z-domain (Affibody), adnectin, lipocalin, affilin,anticalin, knottin, fynomer, atrimer, kunitz domain (see, e.g., WO2004/063337), CTLA4, or avimer (see, e.g., U.S. Publ. Nos. 2004/0175756,2005/0053973, 2005/0048512, and 2006/0008844).

In additional embodiments, the targeting moiety is an antibody or aderivative of the antigen binding domain of an antibody that hasspecific affinity for an epitope of a cell surface antigen of interestexpressed on the surface of a target cell. In some embodiments, thetargeting moiety is a full-length antibody. In some embodiments, thetargeting moiety is an antigen binding portion of an antibody. In someembodiments, the targeting moiety is an scFv. In other embodiments, thetargeting moiety is a Fab. In some embodiments, the targeting moietycomprises a binding domain derived from the antigen binding domain of anantibody (e.g., an scFv, Fab, Fab′, F(ab′)2, an Fv fragment, adisulfide-linked Fv (sdFv), a Fd fragment containing VH and CH1 domains,an scFv, a minibody, a BiTE, a Tandab, a diabody ((VL-VH)₂ or (VH-VL)₂),a single domain antibody (e.g., an sdAb such as a nanobody (either VL orVH)), and a camelid VHH domain). In some embodiments, the targetingmoiety comprises one or more complementarity determining regions (CDRs)of antibody origin. Examples of suitable antibody-based targetingmoieties for the disclosed targeted liposomes include a full-lengthhuman antibody, a humanized antibody, a chimeric antibody, an antigenbinding fragment of an antibody, a single chain antibody, asingle-domain antibody, a bi-specific antibody, a synthetic antibody, apegylated antibody and a multimeric antibody. The antibody of theprovided targeted liposomes can have a combination of the abovecharacteristics. For example, a humanized antibody can be an antigenbinding fragment and can be pegylated and multimerized as well.

The term “humanized antibody” refers to forms of non-human (e.g.,murine) antibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e.g., mouse, rat, rabbit, and hamster) that have the desiredspecificity, affinity, and capability (Jones et al., Nature 321:522-525(1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,Science 239:1534-1536 (1988)). In some instances, the Fv frameworkregion (FR) residues of a human immunoglobulin are replaced with thecorresponding residues in an antibody from a non-human species that hasthe desired specificity, affinity, and capability. The humanizedantibody can be further modified by the substitution of additionalresidues either in the Fv framework region and/or within the replacednon-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDR regionsthat correspond to the non-human immunoglobulin whereas all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. Nos. 5,225,539 and5,639,641.

In further embodiments, the targeting moiety has specific affinity foran epitope of a surface antigen of a target cell of interest. In someembodiments, the target cell is a cancer cell. In some embodiments, thetarget cell is a tumor cell. In other embodiments, the target cell is animmune cell.

In some embodiments, the targeting moiety has specific affinity for anepitope expressed on a tumor cell surface antigen. The term “tumor cellsurface antigen” refers to an antigen that is common to a specifichyperproliferative disorder such as cancer. In some embodiments, thetargeting moiety has specific affinity for an epitope of a tumor cellsurface antigen that is a tumor associated antigen (TAA). A TAA is anantigen that is found on both tumor and some normal cells. A TAA may beexpressed on normal cells during fetal development when the immunesystem is immature and unable to respond or may be normally present atextremely low levels on normal cells but which are expressed at muchhigher levels on tumor cells. Because of the dynamic nature of tumors,in some instances, tumor cells may express unique antigens at certainstages, and at others also express antigens that are also expressed onnon-tumor cells. Thus, inclusion of a certain marker as a TAA does notpreclude it being considered a tumor specific antigen. In someembodiments, the targeting moiety has specific affinity for an epitopeof a tumor cell surface antigen that is a tumor specific antigen (TSA).A TSA is an antigen that is unique to tumor cells and does not occur onother cells in the body. In some embodiments, the targeting moiety hasspecific affinity for an epitope of a tumor cell surface antigenexpressed on the surface of a cancer including but not limited toprimary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer(e.g., NSCLC or SCLC), liver cancer, non-Hodgkin's lymphoma, Hodgkin'slymphoma, leukemias, multiple myeloma, glioblastoma, neuroblastoma,uterine cancer, cervical cancer, renal cancer, thyroid cancer, bladdercancer, kidney cancer, mesothelioma, and adenocarcinomas such as breastcancer, prostate cancer, ovarian cancer, pancreatic cancer, lung cancer,colon cancer and other cancers known in the art In some embodiments, thetargeting moiety has specific affinity for an epitope of a cell surfaceantigen expressed on the surface of a cell in the tumor microenvironment(e.g., and antigen such as VEGFR and TIE1, or TIE2 expressed onendothelial cells and macrophage, respectively, or an antigen expressedon tumor stromal cells such as cancer-associated fibroblasts (CAFs)tumor infiltrating T cells and other leukocytes, and myeloid cellsincluding mast cells, eosinophils, and tumor-associated macrophages(TAM).

In some embodiments, the targeted liposome αPTHF composition (e.g.,TLp-αPTHF or TPLp-αPTHF) comprises a targeting moiety that has specificaffinity for an epitope of a cancer or tumor cell surface antigen thatis preferentially/differentially expressed on a target cell such as acancer cell or tumor cell, compared to normal or non-tumor cells, thatis present on a tumor cell but absent or inaccessible on a non-tumorcell. For example, in some situations, the tumor antigen is on thesurface of both normal cells and malignant cancer cells but the tumorepitope is only exposed in a cancer cell. As a further example, a tumorcell surface antigen may experience a confirmation change in a cancerousstate that causes a cancer cell specific epitope to be present. Atargeting moiety with specific affinity to an epitope on a targetabletumor cell surface antigen described herein or otherwise known in theart is useful and is encompassed by the disclosed compositions andmethods. In some embodiments, the tumor cell with the tumor cell surfaceantigen is a cancer cell. Examples of such tumor cell surface antigensinclude, without limitation folate receptor alpha, folate receptor betaand folate receptor delta.

In further embodiments, the targeting moiety comprises a polypeptidetargeting moiety such as an antibody or an antigen-binding antibodyfragment and the targeting moiety has binding specificity for a folatereceptor. In some embodiments, the targeting moiety binds a folatereceptor with an equilibrium dissociation constant (Kd) in a range of0.5×10⁻¹⁰ to 10×10⁻⁶ as determined using BIACORE® analysis. In someembodiments, the folate receptor bound by the targeting moiety is one ormore folate receptors selected from the group consisting of: folatereceptor alpha (FR-α), folate receptor beta (FR-β), and folate receptordelta (FR-δ). In a further embodiment, the targeting moiety has specificaffinity for at least two antigens selected from the group consisting offolate receptor alpha, folate receptor beta, and folate receptor delta.In another embodiment, the targeting moiety has specific affinity forfolate receptor alpha; folate receptor beta; and folate receptor delta.

In some embodiments, the targeting moiety has a specific affinity for anepitope of a cell surface antigen that internalizes the targeting moietyupon binding. Numerous cell surface antigens that internalize bindingpartners such as antibodies upon binding are known in the art and areenvisioned to be binding targets for the targeting moieties expressed onthe targeted liposome αPTHF compositions (e.g., TLp-αPTHF or TPLp-αPTHF)disclosed herein.

In some embodiments, the targeting moiety has a specific affinity for anepitope of a cell surface antigen selected from the group consisting of:GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin,fibronectin extra-domain B (ED-B), VEGFR2 (CD309), tenascin, collagenIV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, the targetingmoiety has a specific affinity for an epitope on a cell surfaceantigen(s) derived from, or determined to be expressed on, a specificsubject's cancer (tumor) such as a neoantigen.

In some embodiments, the targeting moiety has a specific affinity for anepitope of a cell surface antigen selected from the group consisting ofmannose-6-phosphate receptor, transferrin receptor, and a cell adhesionmolecule (CAM). In further embodiments, the targeting moiety has aspecific affinity for an epitope of a CAM is selected from the groupconsist of: intercellular adhesion molecule (ICAM), platelet-endothelialcell adhesion molecule (PECAM), activated leukocyte cell adhesionmolecule (ALCAM), B-lymphocyte cell adhesion molecule (BL-CAM), vascularcell adhesion molecule (VCAM), mucosal vascular addressin cell adhesionmolecule (MAdCAM), CD44, LFA-2, LFA-3, and basigin.

A discussed herein, folate receptors (FRs) are distinct from reducedfolate carriers (RFCs) and exploit different pathways for bringingfolates and antifolates into cells. In some embodiments, the targetingmoiety specifically binds a folate receptor. In further embodiments, thetargeting moiety specifically binds a folate receptor selected fromfolate receptor alpha, folate receptor beta and folate receptor delta.Antibodies to folate receptor alpha can routinely be generated usingtechniques known in the art. Moreover, the sequences of numerousanti-folate receptor antibodies are in the public domain and/orcommercially available and are readily obtainable.

Murine antibodies against folate receptor are examples of antibodiesthat can be used as targeting moieties of the disclosed targetedliposome is a murine antibody against folate receptor. The sequence ofthese antibodies are known and are described, for example, in U.S. Pat.Nos. 5,646,253; 8,388,972; 8,871,206; and 9,133,275, and Intl. Appl.Nos. PCT/US2011/056966, and PCT/US2012/046672. For example, based on thesequences already in the public domain, the gene for the antibodies canbe synthesized and placed into a transient expression vector and theantibody was produced in HEK-293 transient expression system. Theantibody can be a complete antibody, a Fab, or any of the variousantibody variations discussed herein or otherwise known in the art.

In some embodiments, the targeted liposome (e.g., TL-αPTHF or TPL-αPTHF)contains from 1 to 1,000, 30-1,000, 50-1,000, or more than 1,000,targeting moieties on its surface. In some embodiments, the targetedliposome contains from 30 to 500, 30 to 250 or 30-200, targetingmoieties, or any range therein between. In some embodiments, thetargeted liposome contains less than 220 targeting moieties, less than200 targeting moieties, or less than 175 targeting moieties. In someembodiments, the targeting moiety is non-covalently bonded to theoutside of the liposome (e.g., via ionic interaction or a GPI anchor).

In some embodiments, the molecules on the outside of the targetedliposome (e.g., TL-αPTHF or TPL-αPTHF) include a lipid, a targetingmoiety, a steric stabilizer (e.g., a PEG), a maleimide, and acholesterol. In some embodiments, the targeting moiety is covalentlybound via a maleimide functional group. In some embodiments, thetargeting moiety is covalently bound to a liposomal component or asteric stabilizer such as a PEG molecule. In some embodiments, all thetargeting moieties of the liposome are bound to one component of theliposome such as a PEG. In other embodiments, the targeting moieties ofthe targeted liposome are bound to different components of the liposome.For example, some targeting moieties may be bound to the lipidcomponents or cholesterol, some targeting moieties may be bound to thesteric stabilizer (e.g., PEG) and still other targeting moieties may bebound to a detectable marker or to another targeting moiety. In someembodiments, the outside of the targeted liposome (e.g., TL-αPTHF orTPL-αPTHF) further comprises one or more of an immunostimulatory agent,a detectable marker and a maleimide disposed on at least one of the PEGand the exterior of the liposome.

In some embodiments, the targeted liposome (e.g., TL-αPTHF or TPL-αPTHF)is anionic or neutral. In some embodiments, the targeted anionic orneutral liposome has a diameter in the range of 20 nm to 500 nm or 20 nmto 200 nm, or any range therein between. In further embodiments, thetargeted anionic or neutral liposome has a diameter in the range of 80nm to 120 nm, or any range therein between.

In other embodiments, the targeted liposome (e.g., TL-αPTHF orTPL-αPTHF) is cationic. In some embodiments, the targeted anionic orneutral liposome has a diameter in the range of 20 nm to 500 nm or 20 nmto 200 nm, or any range therein between. In further embodiments, thetargeted anionic or neutral liposome has a diameter in the range of 80nm to 120 nm, or any range therein between.

In additional embodiments, the liposomal composition comprises targetedliposomes (e.g., TL-αPTHF or TPL-αPTHF) and 30-70%, 30-60%, or 30-50%,w/w of the polyglutamated alpha THF, or any range therein between. Insome embodiments, the targeted liposomes comprise at least 1%, 5%, 10%,15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or morethan 75%, w/w of the polyglutamated alpha THF. In some embodiments,during the process of preparing the targeted liposomes, at least 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, ormore than 75%, of the starting material of polyglutamated alpha THF isencapsulated (entrapped) in the targeted liposomes.

In some embodiments, the targeted liposomal compositions comprise30-70%, 30-60%, or 30-50%, w/w of the tetraglutamated alpha THF, or anyrange therein between In some embodiments, the targeted liposomescomprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the tetraglutamatedalpha THF. In some embodiments, during the process of preparing thetargeted liposomes, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, of the startingmaterial of tetraglutamated alpha THF is encapsulated (entrapped) in thetargeted liposomes.

In some embodiments, the targeted liposomal compositions comprise30-70%, 30-60%, or 30-50%, w/w of the pentaglutamated alpha THF, or anyrange therein between In some embodiments, the targeted liposomescomprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the pentaglutamatedalpha THF. In some embodiments, during the process of preparing thetargeted liposomes, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, of the startingmaterial of pentaglutamated alpha THF is encapsulated (entrapped) in thetargeted liposomes.

In some embodiments, the targeted liposomal compositions comprise30-70%, 30-60%, or 30-50%, w/w of the hexaglutamated alpha THF, or anyrange therein between In some embodiments, the targeted liposomescomprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the hexaglutamatedalpha THF. In some embodiments, during the process of preparing thetargeted liposomes, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, of the startingmaterial of hexaglutamated alpha THF is encapsulated (entrapped) in thetargeted liposomes.

Methods and techniques for covalently associating polypeptide targetingmoieties with a liposome surface molecule are known in the art and canreadily be applied to prepare the TL-αPTHF or TPL-αPTHF liposomecompositions.

Chemical binding of non-proteinaceous targeting moieties and othercompositions to the liposomal surface may be employed. Thus, anon-proteinaceous moiety, may be covalently or non-covalently linked to,embedded or adsorbed onto the liposome using any linking or bindingmethod and/or any suitable chemical linker known in the art. The exacttype and chemical nature of such cross-linkers and cross linking methodsis preferably adapted to the type of affinity group used and the natureof the liposome. Methods for binding or adsorbing or linking thetargeting moiety are also well known in the art. For example, in someembodiments, the targeting moiety may be attached to a group at theinterface via, but not limited to, polar groups such as amino, SH,hydroxyl, aldehyde, formyl, carboxyl, His-tag or other polypeptides. Inaddition, the targeting moiety may be attached via, but not limited to,active groups such as succinimidyl succinate, cyanuric chloride, tosylactivated groups, imidazole groups, CNBr, NHS, Activated CH, ECH, EAH,Epoxy, Thiopropyl, Activated Thiol, etc., Moreover, the targeting moietymay be attached via, but not limited to, hydrophobic bonds (Van DerWaals) or electrostatic interactions that may or may not includecross-linking agents (e.g., bivalent anions, poly-anions, poly-cationsetc.).

(4) Manufacture of Liposomes

In some embodiments, the disclosure provides a method of making aliposomal composition disclosed herein. In one embodiment, the methodincludes forming a mixture comprising: (1) a liposomal component; and(2) a polyglutamated alpha (e.g., pentaglutamated or hexaglutamated)tetrahydrofolate in aqueous solution. In further embodiments, themixture comprises a pegylated liposomal component. The mixture is thenhomogenized to form liposomes in the aqueous solution. Further, themixture can be extruded through a membrane to form liposomes enclosingthe αPTHF in an aqueous solution. It is understood the liposomalcomponents of this disclosure can comprise any lipid (includingcholesterol) including functionalized lipids and lipids attached totargeting moieties, detectable labels, and steric stabilizers, or anysubset of all of these. It is further noted that the bioactive αPTHF inaqueous solution can comprise any reagents and chemicals discussedherein or otherwise known in the art for the interior or exterior of theliposome including, for example, buffers, salts, and cryoprotectants.

In some embodiments, the disclosure provides a method of making atargeted pegylated liposomal αPTHF (targeted-PLp-αPTHF) or non-targetedPLp-αPTHF disclosed herein. In one embodiment, the method includesforming a mixture comprising: (1) a liposomal component; (2) apolyglutamated alpha (e.g., pentaglutamated or hexaglutamated)tetrahydrofolate in aqueous solution; and (3) the targeting moiety. Themixture is then homogenized to form liposomes in the aqueous solution.Further, the mixture may be extruded through a membrane to formliposomes enclosing the targeted αPTHF in an aqueous solution. It isunderstood that the targeted pegylated liposomal components can compriseany lipid (including cholesterol) including functionalized lipids andlipids attached to targeting moieties, detectable labels, and stericstabilizers, or any subset of all of these. It is further noted that thetargeted pegylated liposome can comprise any reagents and chemicalsdiscussed herein or otherwise known in the art for the interior orexterior of the liposome including, for example, buffers, salts, andcryoprotectants.

The above methods optionally further comprise the step of lyophilizingthe composition after the removing step to form a lyophilizedcomposition. As stated above, targeted-PTPLA or non-targeted-PTPLA inaqueous solution may comprise a cryoprotectant described herein orotherwise known in the art. If the composition is to be lyophilized, acryoprotectant may be preferred.

Additionally, after the lyophilizing step, the method optionally furthercomprises the step of reconstituting the lyophilized composition bydissolving the composition in a solvent after the lyophilizing step.Methods of reconstitution are known in the art. One preferred solvent iswater. Other preferred solvents include saline solutions and bufferedsolutions.

While certain exemplary embodiments, are discussed herein, it isunderstood that liposomes can be made by any method that is known in theart. See, for example, G. Gregoriadis (editor), Liposome Technology,vol. 1-3, 1st edition, 1983; 2nd edition, 1993, CRC Press, 45 BocaRaton, Fla. Examples of methods suitable for making liposomecompositions include extrusion, reverse phase evaporation, sonication,solvent (e.g., ethanol) injection, microfluidization, detergentdialysis, ether injection, and dehydration/rehydration. The size ofliposomes can routinely be controlled by controlling the pore size ofmembranes used for low pressure extrusions or the pressure and number ofpasses utilized in microfluidization or any other suitable methods knownin the art.

In general, the αPTHF is contained inside, that is, in the inner(interior) space of the liposomes. In one embodiment, substitutedammonium is partially or substantially completely removed from the outermedium surrounding the liposomes. Such removal can be accomplished byany suitable means known in the art (e.g., dilution, ion exchangechromatography, size exclusion chromatography, dialysis,ultrafiltration, and precipitation). Accordingly, the methods of makingliposomal compositions set forth above or otherwise known in the art canoptionally further comprise the step of removing αPTHF in aqueoussolution outside of the liposomes after forming the liposomes, forexample, by the homogenization or by the extruding step.

In other embodiments, the disclosure provides a targeted pegylatedliposomal αPTHF (TPLp-αPTHF) that selectively targets folate receptorscomprising: a liposome including an interior space, a αPTHF disposedwithin the interior space, a steric stabilizer molecule attached to anexterior of the liposome, and a targeting moiety comprising a proteinwith specific affinity for at least one folate receptor, said targetingmoiety attached to at least one of the steric stabilizer and theexterior of the liposome. The components of this embodiment, may be thesame as described for other embodiments, of this disclosure. Forexample, the targeted pegylated liposomal αPTHF and the stericstabilizer which may be PEG, are as described in other parts of thisdisclosure.

In some embodiments, the disclosure provides a method of preparing atargeted composition comprising a pegylated liposome including anentrapped and/or encapsulated αPTHF; a targeting moiety an amino acidchain, the amino acid chain comprising a plurality of amino acids, thetargeting moiety having a specific affinity for at least one type offolate receptor, the specific affinity being defined to include anequilibrium dissociation constant (Kd) in a range of 0.5×10⁻¹⁰ to10×10⁻⁶ moles [0.05 nM to 10 μM] for at least one type folate receptor,the targeting moiety attached to one or both of a PEG and an exterior ofthe liposome, the method comprising: forming a mixture comprising:liposomal components and αPTHF in solution; homogenizing the mixture toform liposomes in the solution; processing the mixture to form liposomesentrapping and/or encapsulating αPTHF; and providing a targeting moietyon a surface of the liposomes entrapping and/or encapsulating the αPTHF,the targeting moiety having specific affinity for at least one of folatereceptor alpha (FR-α), folate receptor beta (FR-β) and folate receptordelta (FR-δ). In some embodiments, the method comprising: forming amixture comprising: liposomal components and alpha polyglutamatedtetrahydrofolate in solution; forming liposomes entrapping and/orencapsulating alpha polyglutamated tetrahydrofolate, for example byhomogenizing or otherwise processing the mixture to form liposomes; andproviding a targeting moiety on a surface of the liposomes entrappingand/or encapsulating the alpha polyglutamated tetrahydrofolate, thetargeting moiety having specific affinity for at least one of folatereceptor alpha (FR-α), folate receptor beta (FR-β) and folate receptordelta (FR-δ). In some embodiments, the processing includes one or moreof: thin film hydration, extrusion, in-line mixing, ethanol injectiontechnique, freezing-and-thawing technique, reverse-phase evaporation,dynamic high pressure microfluidization, microfluidic mixing, doubleemulsion, freeze-dried double emulsion, 3D printing, membrane contactormethod, and stirring, and once the particles have been formed, theparticles can have their sizes further modified by one or more ofextrusion and sonication. In some embodiments, during the process ofpreparing the liposomes at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, of thestarting material of polyglutamated alpha THF is encapsulated(entrapped) in the targeted liposomes. In some embodiments, theliposomes are anionic or neutral. In some embodiments, the targetingmoiety has the specific affinity for one or more of: folate receptoralpha (FR-α), folate receptor beta (FR-β) and folate receptor delta(FR-δ). In further embodiments, the targeting moiety has the specificaffinity for folate receptor alpha (FR-α) and folate receptor beta(FR-β). In additional embodiments, the targeting moiety has the specificaffinity for an epitope on a tumor cell surface antigen that is presenton a tumor cell but absent or inaccessible on a non-tumor cell.

Liposomes can also be prepared to target particular cells, organs, orcell organelles by varying phospholipid composition or by insertingreceptors or counter-receptors into the liposomes. For example,liposomes, prepared with a high content of a nonionic surfactant, havebeen used to target the liver. (See, e.g., Japanese Patent 04-244,018 toHayakawa et al.; Kato et al., Biol. Pharm. Bull. 16:960, (1993.)) Aliposomal formulation of dipalmitoylphosphatidylcholine (DPPC) with asoybean-derived sterylglucoside mixture (SG) and cholesterol (Ch) hasalso been shown to target the liver. (See Shimizu et al., Biol. Pharm.Bull. 20:881, 1997.)

B. Antibody Delivery Vehicles

In additional embodiments, the disclosure provides an antibody deliveryvehicle (e.g., an immunoconjugate (ADC)). In some embodiments, thedisclosure provides an immunoconjugate having the formula(A)-(L)-(αPTHF), wherein: (A) is an antibody or antigen binding fragmentof an antibody; (L) is a linker; and (αPTHF) is a αPTHF compositiondescribed herein; and wherein said linker (L) links (A) to (αPTHF).

In some embodiments, the antibody or antigen binding antibody fragmenthas specific affinity for an epitope of a cell surface antigen on a cellof interest (e.g., an epitope and/or antigen described herein). Incertain embodiments, the antibody binds to an antigen target that isexpressed in or on the cell membrane (e.g., on the cell surface) of acancer/tumor and the antibody is internalized by the cell after bindingto the (antigen) target, after which the αPTHF is releasedintracellularly. In some embodiments, the antibody is a full lengthantibody.

The antibody or antigen binding antibody fragment of the (A)-(L)-(αPTHF)immunoconjugate can be an IgA, IgD, IgE, IgG or IgM antibody. Thedifferent classes of immunoglobulins have different and well knownsubunit structures and three-dimensional configurations. In certainembodiments, the antibody is an IgG antibody. In some embodiments, theantibody is an IgG1, IgG2, IgG3 or IgG4 antibody. In certainembodiments, the antibody is an IgG1 antibody.

In some embodiments, (A) is an antigen binding fragment of an antibody.In some embodiments, (A) is an antigen binding fragment of an antibody.

A “linker” is any chemical moiety that is capable of linking a compound,usually a drug, such as a αPTHF, to an antibody or antigen bindingfragment of an antibody in a stable, covalent manner. The linkers can besusceptible to or be substantially resistant to acid-induced cleavage,light-induced cleavage, peptidase-induced cleavage, esterase-inducedcleavage, and disulfide bond cleavage, at conditions under which thecompound or the antibody remains active. Suitable linkers are known inthe art and include, for example, disulfide groups, thioether groups,acid labile groups, photolabile groups, peptidase labile groups andesterase labile groups. Linkers also include charged linkers, andhydrophilic forms thereof.

In some embodiments, the linker is selected from the group consisting ofa cleavable linker, a non-cleavable linker, a hydrophilic linker, and adicarboxylic acid based linker. In another embodiment, the linker is anon-cleavable linker. In another embodiment, the linker is selected fromthe group consisting: N-succinimidyl 4-(2-pyridyldithio) pentanoate(SPP); N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) orN-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB);N-succinimidyl 4-(maleimidomethyl) cyclohexane-carboxylate (SMCC);N-sulfosuccinimidyl 4-(maleimidomethyl) cyclohex-anecarboxylate(sulfoSMCC); N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB); andN-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester(NHS-PEG4-ma-leimide). In a further embodiment, the linker isN-succinimidyl-[(N-maleimido-propionamido)-tetraethyleneglycol] ester(NHS-PEG4-maleimide).

In some embodiments, the α polyglutamated THF is attached (coupled) tothe antibody or antigen binding antibody fragment of the immunoconjugatedirectly, or through a linker using techniques known in the art. Suchattachment of one or more αPTHF can include many chemical mechanisms,such as covalent binding, affinity binding, intercalation, coordinatebinding and complexation. Covalent binding of the αPTHF and antibody orantigen binding antibody fragment can be achieved by direct condensationof existing side chains or by the incorporation of external bridgingmolecules. Many bivalent or polyvalent agents are useful in associatingpolypeptides to other proteins with coupling agents such ascarbodiimides, diisocyanates, glutaraldehyde, diazobenzenes, andhexamethylene diamines. This list is not intended to be exhaustive ofthe various coupling agents known in the art but, rather, is exemplaryof the more common coupling agents. In some embodiments, the antibody orantigen binding antibody fragment is derivatized and then attached tothe α polyglutamated THF. Alternatively, the αPTHF can be derivatizedand attached to the antibody or antigen binding antibody fragment usingtechniques known in the art.

In some embodiments, the immunoconjugate comprises an antibody or anantigen-binding fragment of an antibody and αPTHF containing 4, 5, 2-10,4-6, or more than 5, glutamyl groups (including the glutamyl group intetrahydrofolate). In some embodiments, the immunoconjugate comprisesαPTHF that comprises two or more glutamyl groups in the L-form. In otherembodiments, the immunoconjugate comprises αPTHF that comprises aglutamyl group in the D-form. In further embodiments, theimmunoconjugate comprises αPTHF that comprises a glutamyl group in theD-form and two or more glutamyl groups in the L-form. In additionalembodiments, the immunoconjugate comprises an αPTHF that comprises twoor more glutamyl groups that have a gamma carboxyl linkage. In someembodiments, the immunoconjugate comprises tetraglutamated αPTHF. Insome embodiments, the immunoconjugate comprises pentaglutamated αPTHF.In further embodiments, the immunoconjugate comprises L-pentaglutamatedαPTHF, a D-pentaglutamated αPTHF, or an L- and D-pentaglutamated αPTHF.In some embodiments, the immunoconjugate comprises a hexaglutamatedαPTHF (Lp-αPTHF). In further embodiments, the immunoconjugate comprisesan L-hexaglutamated αPTHF, a D-hexaglutamated αPTHF, or an L- andD-hexaglutamated αPTHF.

In some embodiments, the antibody delivery vehicle composition comprisesa αPTHF and an antibody or an antigen binding antibody fragment that hasspecific affinity for an epitope of a cell surface antigen selected fromthe group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, afolate receptor (e.g., folate receptor-α, folate receptor-β or folatereceptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, Nectin 4,ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9(Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, Tissue factor, LIV-1(ZIP6), CGEN-15027, P cadherin, fibronectin extra-domain B (ED-B),VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor,HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6,IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10,SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20,CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56,CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R,IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA1, EphA2, EphA3,EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, anintegrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242 antigen, Apo2,PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1,VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK,ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK. In someembodiments, the delivery vehicle comprises a targeting moiety that hasspecific affinity for an epitope of a cell surface antigen(s) derivedfrom, or determined to be expressed on, a specific subject's cancer(tumor) such as a neoantigen.

In some embodiments, the antibody delivery vehicle composition comprisesa αPTHF and an antibody or an antigen binding antibody fragment that hasspecific affinity for an epitope of an antigen selected from the groupconsisting of mannose-6-phosphate receptor, transferrin receptor, and acell adhesion molecule (CAM). In further embodiments, the targetingmoiety has a specific affinity for an epitope of a CAM is selected fromthe group consist of: intercellular adhesion molecule (ICAM),platelet-endothelial cell adhesion molecule (PECAM), activated leukocytecell adhesion molecule (ALCAM), B-lymphocyte cell adhesion molecule(BL-CAM), vascular cell adhesion molecule (VCAM), mucosal vascularaddressin cell adhesion molecule (MAdCAM), CD44, LFA-2, LFA-3, andbasigin

In some embodiments, the antibody delivery vehicle composition comprises1, 2, 3, 4, 5, 5-10, or greater than 10, glutamyl groups. In someembodiments, the antibody delivery vehicle composition comprises 1, 2,3, 4, 5, 5-10, or greater than 10, glutamyl groups. In some embodiments,the antibody delivery vehicle composition comprises 1, 2, 3, 4, 5, 5-10,or greater than 10, glutamyl groups.

IV. Pharmaceutical Compositions and Administration

In some embodiments, the liposome composition is provided as apharmaceutical composition containing the liposome and a carrier, e.g.,a pharmaceutically acceptable carrier. Examples of pharmaceuticallyacceptable carriers contained in the provided pharmaceuticalcompositions include normal saline, isotonic dextrose, isotonic sucrose,Ringer's solution, and Hanks' solution. In some embodiments, a buffersubstance is added to maintain an optimal pH for storage stability ofthe pharmaceutical composition. In some embodiments, the pH of thepharmaceutical composition is between 6.0 and 7.5. In some embodiments,the pH is between 6.3 and 7.0. In further embodiments, the pH is 6.5.Ideally the pH of the pharmaceutical composition allows for bothstability of liposome membrane lipids and retention of the entrappedentities. Histidine, hydroxyethylpiperazine-ethylsulfonate (HEPES),morpholipoethylsulfonate (MES), succinate, tartrate, and citrate,typically at 2-20 mM concentration, are exemplary buffer substances.Other suitable carriers include, e.g., water, buffered aqueous solution,0.4% NaCl, and 0.3% glycine. Protein, carbohydrate, or polymericstabilizers and tonicity adjusters can be added, e.g., gelatin, albumin,dextran, or polyvinylpyrrolidone. The tonicity of the composition can beadjusted to the physiological level of 0.25-0.35 mol/kg with glucose ora more inert compound such as lactose, sucrose, mannitol, or dextrin.These compositions can routinely be sterilized using conventional,sterilization techniques known in the art (e.g., by filtration). Theresulting aqueous solutions can be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile aqueous medium prior to administration.

The provided pharmaceutical liposome compositions can also contain otherpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, and tonicity adjusting agents, for example, sodium acetate,sodium lactate, sodium chloride, potassium chloride, and calciumchloride. Additionally, the liposome suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as alpha-tocopherol and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

The liposome concentration in the provided fluid pharmaceuticalformulations can vary widely depending upon need, e.g., from less thanabout 0.05% usually or at least about 2-10% to as much as 30-50% byweight and will be selected primarily by fluid volumes, and viscosities,in accordance with the particular mode of administration selected. Forexample, the concentration may be increased to lower the fluid loadassociated with treatment. This may be particularly desirable inpatients having atherosclerosis-associated congestive heart failure orsevere hypertension. Alternatively, liposome pharmaceutical compositionscomposed of irritating lipids may be diluted to low concentrations tolessen inflammation at the site of administration.

Some embodiments, relate to a method of delivering a targeted pegylatedliposomal formulation of αPTHF, to a tumor expressing folate receptor onits surface. An exemplary method comprises the step of administering aliposome pharmaceutical composition provided herein in an amount todeliver a therapeutically effective dose of the targeted pegylatedliposomal αPTHF to the tumor.

The amount of liposome pharmaceutical composition administered willdepend upon the particular αPTHF entrapped inside the liposomes, thedisease state being treated, the type of liposomes being used, and thejudgment of the clinician. Generally the amount of liposomepharmaceutical composition administered will be sufficient to deliver atherapeutically effective dose of the particular therapeutic entity.

The quantity of liposome pharmaceutical composition necessary to delivera therapeutically effective dose can be determined by routine in vitroand in vivo methods, common in the art of drug testing. See, forexample, D. B. Budman, A. H. Calvert, E. K. Rowinsky (editors). Handbookof Anticancer Drug Development, L W W, 2003. Therapeutically effectivedosages for various therapeutic compositions are known to those skilledin the art. In some embodiments, a therapeutic entity delivered via thepharmaceutical liposome composition and provides at least the same orhigher activity than the activity obtained by administering the sameamount of the therapeutic entity in its routine non-liposomeformulation. Typically, the dosages for the liposome pharmaceuticalcomposition is in a range for example, between about 0.005 and about5000 mg of the therapeutic entity per square meter (m²) of body surfacearea, most often, between about 0.1 and about 100 mg therapeutic entityper m² of body surface area.

For example, if the subject has a tumor, an effective amount may be thatamount of the agent (e.g., αPTHF composition) that reduces the tumorvolume or load (as for example determined by imaging the tumor).Effective amounts can also routinely be assessed by the presence and/orfrequency of cancer cells in the blood or other body fluid or tissue(e.g., a biopsy). If the tumor is impacting the normal functioning of atissue or organ, then the effective amount can routinely be assessed bymeasuring the normal functioning of the tissue or organ. In someinstances the effective amount is the amount required to lessen oreliminate one or more, and preferably all, symptoms.

Pharmaceutical compositions comprising the αPTHF compositions (e.g.,liposomes containing a pentaglutamated or hexaglutamatedtetrahydrofolate) are also provided. Pharmaceutical compositions aresterile compositions that comprise a sample liposome and preferablyαPTHF, preferably in a pharmaceutically-acceptable carrier.

Unless otherwise stated herein, a variety of administration routes areavailable. The particular mode selected will depend, upon the particularactive agent selected, the particular condition being treated and thedosage required for therapeutic efficacy. The provided methods can bepracticed using any known mode of administration that is medicallyacceptable and in accordance with good medical practice. In someembodiments, the administration route is an injection. In furtherembodiments, the injection is by a parenteral route elected from anintramuscular, subcutaneous, intravenous, intraarterial,intraperitoneal, intraarticular, intraepidural, intrathecal,intravenous, intramuscular, or intra sternal injection. In someembodiments, the administration route is an infusion. In additionalembodiments, the administration route is oral, nasal, mucosal,sublingual, intratracheal, ophthalmic, rectal, vaginal, ocular, topical,transdermal, pulmonary, or inhalation.

Therapeutic compositions containing αPTHF compositions such as theliposomal αPTHF compositions described herein can be conventionallyadministered intravenously, as by injection of a unit dose, for example.The term “unit dose” when used in reference to a therapeutic compositionprovided herein refers to physically discrete units suitable as unitarydosage for the subject, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect inassociation with the required diluent; e.g., carrier, or vehicle. In aspecific embodiment, therapeutic compositions containing an Adapter areadministered subcutaneously.

In some embodiments, the α-PTHF composition is administered in a mannercompatible with the dosage formulation, and in a therapeuticallyeffective amount. The quantity to be administered depends on the subjectto be treated, capacity of the subject's system to utilize the activeingredient, and degree of therapeutic effect desired. Precise amounts ofactive ingredient required to be administered depend on the judgment ofthe practitioner and are peculiar to each individual. However, suitabledosage ranges for systemic application are disclosed herein and dependon the route of administration. Suitable regimes for administration arealso variable, but are typified by an initial administration followed byrepeated doses at one or more hour intervals by a subsequent injectionor other administration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in vivo therapies are contemplated.

The αPTHF composition are formulated, dosed, and administered in afashion consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular patient being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Thedosage ranges for the administration of αPTHF composition are thoselarge enough to produce the desired effect in which the disease symptomsmediated by the target molecule are ameliorated. The dosage should notbe so large as to cause adverse side effects, such as, hyperviscositysyndromes, pulmonary edema, congestive heart failure, and other adverseside effects known in the art. Generally, the dosage will vary with theage, weight, height, body surface area, state of health (e.g., renal andliver function), condition, sex and extent of the disease in the patientand can routinely be determined by one of ordinary skill in the art. Thedosage can be adjusted by the individual physician in the event of anycomplication.

The dosage schedule and amounts effective for therapeutic andprophylactic uses, i.e., the “dosing regimen”, will depend upon avariety of factors, including the cause, stage and severity of thedisease or disorder, the health, physical status, age of the subjectbeing treated, and the site and mode of the delivery of the αPTHFcomposition. Therapeutic efficacy and toxicity of the αPTHF compositioncan be determined by standard pharmaceutical, pharmacological, andtoxicological procedures in cell cultures or experimental animals. Dataobtained from these procedures can likewise be used in formulating arange of dosages for human use. Moreover, therapeutic index (i.e., thedose therapeutically effective in 50 percent of the population dividedby the dose lethal to 50 percent of the population (ED50/LD50)) canreadily be determined using known procedures. The dosage is preferablywithin a range of concentrations that includes the ED50 with little orno toxicity, and may vary within this range depending on the dosage formemployed, sensitivity of the patient, and the route of administration.

The dosage regimen also takes into consideration pharmacokineticsparameters known in the art, such as, drug absorption rate,bioavailability, metabolism and clearance (see, e.g., Hidalgo-Aragones,J. Steroid Biochem. Mol. Biol. 58:611-617 (1996); Groning et al.,Pharmazie 51:337-341 (1996); Fotherby, Contraception 54:59-69 (1996);and Johnson et al., J. Pharm. Sci. 84:1144-1146 (1995)). It is wellwithin the state of the art for the clinician to determine the dosageregimen for each subject being treated. Moreover, single or multipleadministrations of the αPTHF composition can be administered dependingon the dosage and frequency as required and tolerated by the subject.The duration of prophylactic and therapeutic treatment will varydepending on the particular disease or condition being treated. Somediseases are amenable to acute treatment whereas others requirelong-term, chronic therapy. The αPTHF composition can be administeredserially, or simultaneously with the additional therapeutic agent.

In some embodiments, the αPTHF composition is administered in aliposomal composition at a dose of between 0.005 and 5000 mg of αPTHFper square meter of body surface area, or any range therein between. Infurther embodiments, the αPTHF composition is administered in aliposomal composition at a dose of between 0.1 and 1000 mg αPTHF persquare meter of body surface area, or any range therein between.

In some embodiments, the αPTHF composition is administered in animmunoconjugate composition at a dose of 1 mg/kg to 500 mg/kg, 1 mg/kgto 250 mg/kg, 1 mg/kg to 200 mg/kg, 1 mg/kg to 150 mg/kg, 1 mg/kg to 100mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to 25 mg/kg, 1 mg/kg to 20 mg/kg, 1mg/kg to 15 mg/kg, 1 mg/kg to 10 mg/kg, or 1 mg/kg to 5 mg/kg, or anyrange therein between.

In another embodiment, the αPTHF composition is administered incombination with one or more additional therapeutics.

In some embodiment, the PLp-αPTHF and/or targeted-PLp-αPTHF is preparedas an infusion composition, an injection composition, a parenteralcomposition, or a topical composition. In further embodiments, theinjection includes one or more of: intraperitoneal injection, directintratumor injection, intra-arterial injection, and intravenousinjection, subcutaneous injection, intramuscular injection, delivery viatranscutaneous and intranasal route. In a further embodiment, thePLp-αPTHF and/or targeted-PLp-αPTHF is a liquid solution or asuspension. However, solid forms suitable for solution in, or suspensionin, liquid vehicles prior to injection are also provided herein. In someembodiments, the targeted pegylated liposomal αPTHF composition isformulated as an enteric-coated tablet or gel capsule according tomethods known in the art.

In some embodiments, the targeted pegylated liposomal αPTHF formulationsare administered to a tumor of the central nervous system using a slow,sustained intracranial infusion of the liposomes directly into the tumor(e.g., a convection-enhanced delivery (CED)). See, Saito et al., CancerResearch 64:2572-2579 (2004); Mamot et al., J. Neuro-Oncology 68:1-9(2004). In other embodiments, the formulations are directly applied totissue surfaces. Sustained release, pH dependent release, and otherspecific chemical or environmental condition-mediated releaseadministration of the pegylated liposomal αPTHF formulations (e.g.,depot injections and erodible implants) are also provided. Examples ofsuch release-mediating compositions are further described herein orotherwise known in the art.

For administration by inhalation, the compositions can be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,ichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount.

When it is desirable to deliver the compositions systemically, they canformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection can bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers. Pharmaceutical parenteral formulations include aqueoussolutions of the ingredients. Aqueous injection suspensions can containsubstances which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. Alternatively,suspensions of liposomes can be prepared as oil-based suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides.

Alternatively, the non-targeted or targeted pegylated liposomal αPTHFcan be in powder form or lyophilized form for constitution with asuitable vehicle, e.g., sterile pyrogen-free water, before use.

The provided compositions (e.g., αPTHF and liposomes containing theαPTHF) can also be formulated in rectal or vaginal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

Methods of Use and Treatment

In additional embodiments, the disclosure provides methods of usingαPTHF (αPTHF) compositions. In some embodiments, the alpha αPTHFcompositions (e.g., a αPTHF disclosed herein) are used to treat adisease or disorder.

In some embodiments, the disclosure provides a method of killing a cellthat comprises contacting the cell with a composition comprising apolyglutamated alpha tetrahydrofolate (e.g., a αPTHF disclosed herein).In some embodiments, the αPTHF is selected from: (a) polyglutamated5-formyl-THF (e.g., polyglutamated [6S]-5-formyl-THF); (b)polyglutamated 10-formyl-THF (e.g., polyglutamated [6R]-10-formyl-THF);(c) polyglutamated 5,10-methenyl-THF (e.g., polyglutamated[6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF (e.g.,polyglutamated [6S]-5-methyl-THF); (e) polyglutamated TetrahydrofolateTHF (e.g., polyglutamated [6S]-Tetrahydrofolate THF); (f) polyglutamated5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and(g) polyglutamated 5-formimino-THF (e.g., polyglutamated[6S]-5-formimino-THF). In some embodiments, the αPTHF comprisespolyglutamated 5,10-methylene-THF. In further embodiments, the αPTHFcomprises polyglutamated [6R]-5,10-methylene-THF. In other embodiments,the αPTHF comprises polyglutamated [6R,S]-5,10-methylene-THF. In someembodiments, the αPTHF comprises polyglutamated 5-methyl-THF. In furtherembodiments, the αPTHF comprises [6S]-5-methyl-THF. In otherembodiments, the αPTHF comprises [6R,S]-5-methyl-THF. In someembodiments, the αPTHF comprises polyglutamated 5-formyl-THF. In furtherembodiments, the αPTHF comprises polyglutamated [6S]-5-formyl-THF. Inother embodiments, the αPTHF comprises polyglutamated[6R,S]-5-formyl-THF. In some embodiments, the contacted cell is amammalian cell. In further embodiments, the contacted cell is a humancell. In some embodiments, the contacted cell is a hyperproliferativecell. In further embodiments, the hyperproliferative cell is a cancercell. In yet further embodiments, the cancer cell is a primary cell or acell from a cell line obtained/derived from a cancer selected from thegroup consisting of: a non-hematologic malignancy including such as forexample, lung cancer, pancreatic cancer, breast cancer, ovarian cancer,prostate cancer, head and neck cancer, gastric cancer, gastrointestinalcancer, colorectal cancer, esophageal cancer, cervical cancer, livercancer, kidney cancer, biliary duct cancer, gallbladder cancer, bladdercancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervoussystem cancer, and melanoma; and a hematologic malignancy such as forexample, a leukemia, a lymphoma and other B cell malignancies, myelomaand other plasma cell dysplasias or dyscrasias. In yet furtherembodiments, the cancer cell is a primary cell or a cell from a cellline obtained/derived from a cancer selected from: colorectal cancer,breast cancer, gastric cancer (e.g., stomach cancer), pancreatic cancer,liver cancer, lung cancer (e.g., non-small cell lung cancer and/oradenocarcinoma), head and neck cancer, ovarian cancer, gallbladdercancer, and basal cell cancer. In particular embodiments, the cancercell is a primary cell or a cell from a cell line obtained/derived fromcolorectal cancer. In some embodiments, the method is performed in vivo.In other embodiments, the method is performed in vitro. In someembodiments, the αPTHF composition contains 4, 5, 2-10, 4-6, or morethan 5, glutamyl groups. In some embodiments, the αPTHF compositioncomprises pentaglutamated alpha tetrahydrofolate. In some embodiments,the αPTHF composition comprises hexaglutamated alpha tetrahydrofolate.In some embodiments, the αPTHF composition comprises L polyglutamatedalpha tetrahydrofolate. In some embodiments, the αPTHF compositioncomprises D polyglutamated alpha tetrahydrofolate. In some embodiments,the αPTHF composition comprises L and D polyglutamated alphatetrahydrofolate.

In additional embodiments, the disclosure provides a method of killing acell that comprises contacting the cell with a liposome containing αPTHF(e.g., an Lp-αPTHF such as, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF,TLp-αPTHF or TPLp-αPTHF disclosed herein). In some embodiments, theliposome is pegylated (e.g., PLp-αPTHF and NTPLp-αPTHF). In someembodiments, the liposome comprises a targeting moiety on its surfacethat has specific affinity for an epitope of an antigen on the surfaceof the cell (e.g., TLp-αPTHF and TPLp-αPTHF). In further embodiments,the liposome is pegylated and comprises a targeting moiety on itssurface that specifically binds an antigen on the surface of the cell(e.g., TPLp-αPTHF). In some embodiments, the liposome is not pegylated(e.g., PLp-αPTHF and NTPLp-αPTHF). In some embodiments, the unpegylatedliposome comprises a targeting moiety on its surface that specificallybinds an antigen on the surface of the cell (e.g., TLp-αPTHF andTPLp-αPTHF). In some embodiments, the contacted cell is a mammaliancell. In further embodiments, the contacted cell is a human cell. Inadditional embodiments, the contacted cell is a hyperproliferative cell.In further embodiments, the hyperproliferative cell is a cancer cell. Infurther embodiments, the contacted cancer cell is a primary cell or acell from a cell line obtained/derived from a cancer selected from thegroup consisting of: lung cancer (e.g., non-small cell), pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, head and neckcancer, gastric cancer, gastrointestinal cancer, colorectal cancer,esophageal cancer, cervical cancer, liver cancer, kidney cancer, biliaryduct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, melanoma,myeloma, a leukemia and a lymphoma. In some embodiments, the contactedcancer cell is a primary cell or a cell from a cell lineobtained/derived from a cancer selected from the group consisting of:colorectal cancer, breast cancer, gastric cancer (e.g., stomach cancer),pancreatic cancer, liver cancer, lung cancer (e.g., non-small cell lungcancer and/or adenocarcinoma), head and neck cancer, ovarian cancer,gallbladder cancer, and basal cell cancer. In particular embodiments,the contacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from colorectal cancer. In some embodiments, theliposome contains a αPTHF containing 4, 5, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, the liposome comprises a αPTHFselected from: (a) polyglutamated 5-formyl-THF (e.g., polyglutamated[6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF (e.g.,polyglutamated [6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF(e.g., polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF); (e)polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, the liposome comprises polyglutamated 5,10-methylene-THF.In further embodiments, the liposome comprises polyglutamated[6R]-5,10-methylene-THF. In other embodiments, the liposome comprisespolyglutamated [6R,S]-5,10-methylene-THF. In some embodiments, theliposome comprises polyglutamated 5-methyl-THF. In further embodiments,the liposome comprises [6S]-5-methyl-THF. In other embodiments, theliposome comprises [6R,S]-5-methyl-THF. In some embodiments, theliposome comprises polyglutamated 5-formyl-THF. In further embodiments,the liposome comprises polyglutamated [6S]-5-formyl-THF. In otherembodiments, the liposome comprises polyglutamated [6R,S]-5-formyl-THF.In some embodiments, liposome comprises polyglutamated alphatetrahydrofolate. In some embodiments, the liposome comprisestetraglutamated alpha tetrahydrofolate. In some embodiments, theliposome comprises pentaglutamated tetrahydrofolate. In otherembodiments, the liposome comprises alpha hexaglutamatedtetrahydrofolate.

In some embodiments, the disclosure provides a method of killing ahyperproliferative cell that comprises contacting a hyperproliferativecell with a delivery vehicle (e.g., a liposome or antibodyimmunoconjugate) comprising αPTHF (e.g., a αPTHF disclosed herein). Insome embodiments, the delivery vehicle is an antibody (e.g., afull-length IgG antibody, a bispecific antibody, or a scFv). In someembodiments, the delivery vehicle is a liposome (e.g., an Lp-αPTHF suchas, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF)). Insome embodiments, the delivery vehicle is non-targeted. In otherembodiments, the delivery vehicle is targeted and comprises a targetingmoiety on its surface that has specific affinity for an epitope of anantigen on the surface of the hyperproliferative cell. In furtherembodiments, the delivery vehicle comprises a targeting moiety that hasspecific affinity for an epitope of an antigen on the surface of thehyperproliferative cell and wherein the antigen is selected from thegroup consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folatereceptor (e.g., folate receptor-α, folate receptor-β or folatereceptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, Nectin 4,ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9(Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, P cadherin, fibronectin extra-domain B (ED-B),VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor,HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6,IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10,SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20,CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56,CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R,IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA1, EphA2, EphA3,EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, anintegrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242 antigen, Apo2,PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1,VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK,ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK. In someembodiments, the delivery vehicle comprises a targeting moiety that hasspecific affinity for an epitope on a cell surface antigen(s) derivedfrom, or determined to be expressed on, a specific subject's cancer(tumor) such as a neoantigen. In some embodiments, the method isperformed in vivo. In some embodiments, the method is performed invitro. In some embodiments, the delivery vehicle comprises a αPTHFcontaining 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the delivery vehicle comprises a αPTHF αPTHF selected from:(a) polyglutamated 5-formyl-THF (e.g., polyglutamated[6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF (e.g.,polyglutamated [6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF(e.g., polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF); (e)polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, the delivery vehicle comprises polyglutamated5,10-methylene-THF. In further embodiments, the delivery vehiclecomprises polyglutamated [6R]-5,10-methylene-THF. In other embodiments,the delivery vehicle comprises polyglutamated [6R,S]-5,10-methylene-THF.In some embodiments, the delivery vehicle comprises polyglutamated5-methyl-THF. In further embodiments, the delivery vehicle comprises[6S]-5-methyl-THF. In other embodiments, the delivery vehicle comprises[6R,S]-5-methyl-THF. In some embodiments, the delivery vehicle comprisespolyglutamated 5-formyl-THF. In further embodiments, the deliveryvehicle comprises polyglutamated [6S]-5-formyl-THF. In otherembodiments, the delivery vehicle comprises polyglutamated[6R,S]-5-formyl-THF. In some embodiments, the delivery vehicle comprisestetraglutamated alpha tetrahydrofolate. In some embodiments, thedelivery vehicle comprises pentaglutamated alpha tetrahydrofolate. Inother embodiments, the delivery vehicle comprises alpha hexaglutamatedtetrahydrofolate.

In particular embodiments, the method of a killing a hyperproliferativecell is performed using a liposome delivery vehicle that comprises αPTHF(e.g., an Lp-αPTHF such as, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF,TLp-αPTHF or TPLp-αPTHF disclosed herein). In some embodiments, thedelivery vehicle is a non-targeted liposome. In some embodiments, thedelivery vehicle comprises a targeting moiety on its surface that hasspecific affinity for an epitope of an antigen on the surface of thehyperproliferative cell (e.g., TLp-αPTHF and TPLp-αPTHF). In someembodiments the delivery vehicle is a liposome comprising a targetingmoiety on its surface that has specific affinity for an epitope of anantigen on the surface of the hyperproliferative cell (e.g., TLp-αPTHFand TPLp-αPTHF). In some embodiments, the delivery vehicle is a liposomecomprising a targeting moiety on its surface that has specific affinityfor an epitope of an antigen on the surface of the hyperproliferativecell. In further embodiments, the targeting moiety has specific affinityfor an epitope of an antigen selected from the group consisting of:GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin,fibronectin extra-domain B (ED-B), VEGFR2 (CD309), tenascin, collagenIV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, the targetingmoiety has specific affinity for an epitope on a cell surface antigen(s)derived from, or determined to be expressed on, a specific subject'scancer (tumor) such as a neoantigen. In some embodiments, the liposomeis pegylated (e.g., PLp-αPTHF, and NTPLp-αPTHF). In further embodiments,the liposome is pegylated and comprises a targeting moiety on itssurface that has specific affinity for an epitope of an antigen on thesurface of the hyperproliferative cell (e.g., TPLp-αPTHF). In otherembodiments, the embodiments, the liposome is unpegylated. In someembodiments, the liposome is unpegylated and the liposome comprises atargeting moiety on its surface that has specific affinity for anepitope of an antigen on the surface of the hyperproliferative cell(e.g., TPLp-αPTHF). In some embodiments, the liposome comprises a αPTHFcontaining 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the liposome comprises tetraglutamated alphatetrahydrofolate. In some embodiments, the liposome comprisespentaglutamated alpha tetrahydrofolate. In other embodiments, theliposome comprises hexaglutamated alpha tetrahydrofolate. In someembodiments, the liposome comprises a αPTHF selected from: (a)polyglutamated 5-formyl-THF (e.g., polyglutamated [6S]-5-formyl-THF);(b) polyglutamated 10-formyl-THF (e.g., polyglutamated[6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF (e.g.,polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF(e.g., polyglutamated [6S]-5-methyl-THF); (e) polyglutamatedTetrahydrofolate THF (e.g., polyglutamated [6S]-Tetrahydrofolate THF);(f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated[6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g.,polyglutamated [6S]-5-formimino-THF). In some embodiments, the liposomecomprises polyglutamated 5,10-methylene-THF. In further embodiments, theliposome comprises polyglutamated [6R]-5,10-methylene-THF. In otherembodiments, the liposome comprises polyglutamated[6R,S]-5,10-methylene-THF. In some embodiments the liposome comprisespolyglutamated 5-methyl-THF. In further embodiments the liposomecomprises [6S]-5-methyl-THF. In other embodiments, the liposomecomprises [6R,S]-5-methyl-THF. In some embodiments, the liposomecomprises polyglutamated 5-formyl-THF. In further embodiments, theliposome comprises polyglutamated [6S]-5-formyl-THF. In otherembodiments, the liposome comprises polyglutamated [6R,S]-5-formyl-THF.

In additional embodiments, the disclosure provides a method ofinhibiting the proliferation of a cancer cell that comprises contactingthe cancer cell with a delivery vehicle (e.g., a liposome or antibody)comprising αPTHF (e.g., a αPTHF disclosed herein). In some embodiments,the delivery vehicle is an antibody (e.g., a full-length IgG antibody, abispecific antibody, or a scFv). In some embodiments, the deliveryvehicle is a liposome (e.g., an Lp-αPTHF such as, PLp-αPTHF, NTLp-αPTHF,NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF)). In some embodiments, thedelivery vehicle is non-targeted. In some embodiments, the deliveryvehicle is targeted and comprises a targeting moiety on its surface thathas specific affinity for an epitope of an antigen on the surface of thecancer cell. In further embodiments, the delivery vehicle comprises atargeting moiety that has specific affinity for an epitope of a cellsurface antigen selected from the group consisting of: GONMB, TACSTD2(TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-α,folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1,mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b,CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16,Tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin, fibronectinextra-domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin,endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2,FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7,FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15,CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38,CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138,cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA1,EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3,EphB4, EphB6, an integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA,c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB,TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the delivery vehicle comprises a targeting moietythat has specific affinity for an epitope on a cell surface antigen(s)derived from, or determined to be expressed on, a specific subject'scancer (tumor) such as a neoantigen. In some embodiments, the deliveryvehicle is an antibody that has specific affinity for an epitope of anantigen on the surface of the cancer cell. In some embodiments, thecontacted cancer cell is a mammalian cell. In further embodiments, thecontacted cancer cell is a human cell. In additional embodiments, thecontacted cancer cell is a primary cell or a cell from a cell lineobtained/derived from a cancer selected from the group consisting of:lung cancer (e.g., non-small cell), pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, head and neck cancer, gastric cancer,gastrointestinal cancer, colorectal cancer, esophageal cancer, cervicalcancer, liver cancer, kidney cancer, biliary duct cancer, gallbladdercancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer,central nervous system cancer, melanoma, myeloma, a leukemia and alymphoma. In some embodiments, the contacted cancer cell is a primarycell or a cell from a cell line obtained/derived from a cancer selectedfrom the group consisting of: colorectal cancer, breast cancer, gastriccancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lungcancer (e.g., non-small cell lung cancer and/or adenocarcinoma), headand neck cancer, ovarian cancer, gallbladder cancer, and basal cellcancer. In particular embodiments, the contacted cancer cell is aprimary cell or a cell from a cell line obtained/derived from colorectalcancer. In some embodiments, the method is performed in vivo. In someembodiments, the method is performed in vitro. In some embodiments, thedelivery vehicle is an antibody that has specific affinity for anepitope on one of the above-listed cell surface antigens. In otherembodiments, the targeting vehicle is a liposome that comprises atargeting moiety that has specific affinity for an epitope on thesurface of the cancer cell. In other embodiments, the targeting vehicleis a liposome that comprises a targeting moiety that has specificaffinity for an epitope on one of the above-listed cell surfaceantigens. In some embodiments, the delivery vehicle is a liposome thatis pegylated. In other embodiments, the delivery vehicle is a liposomethat is unpegylated. In some embodiments, the delivery vehicle comprisesa αPTHF composition containing 4, 5, 2-10, 4-6, or more than 5, glutamylgroups. In some embodiments, the delivery vehicle comprisestetraglutamated alpha tetrahydrofolate. In some embodiments, thedelivery vehicle comprises pentaglutamated alpha tetrahydrofolate. Inother embodiments, the delivery vehicle comprises hexaglutamated alphatetrahydrofolate. In some embodiments, the delivery vehicle comprises aαPTHF selected from: (a) polyglutamated 5-formyl-THF (e.g.,polyglutamated [6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF(e.g., polyglutamated [6R]-10-formyl-THF); (c) polyglutamated5,10-methenyl-THF (e.g., polyglutamated [6R]-5,10-methenyl-THF); (d)polyglutamated 5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF);(e) polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, the delivery vehicle comprises polyglutamated5,10-methylene-THF. In further embodiments, the delivery vehiclecomprises polyglutamated [6R]-5,10-methylene-THF. In other embodiments,the delivery vehicle comprises polyglutamated [6R,S]-5,10-methylene-THF.In some embodiments, the delivery vehicle comprises polyglutamated5-methyl-THF. In further embodiments, the delivery vehicle comprises[6S]-5-methyl-THF. In other embodiments, the delivery vehicle comprises[6R,S]-5-methyl-THF. In some embodiments, the delivery vehicle comprisespolyglutamated 5-formyl-THF. In further embodiments, the deliveryvehicle comprises polyglutamated [6S]-5-formyl-THF. In otherembodiments, the delivery vehicle comprises polyglutamated[6R,S]-5-formyl-THF.

In further embodiments, the disclosure provides a method of inhibitingthe proliferation of a cancer cell that comprises contacting the cancercell with a liposome comprising αPTHF (e.g., a αPTHF disclosed herein).In some embodiments, the liposome is non-targeted. In some embodiments,the liposome is targeted and comprises a targeting moiety on its surfacethat has specific affinity for an epitope of an antigen on the surfaceof the cancer cell. In further embodiments, the liposome comprises atargeting moiety that has specific affinity for an epitope of a cellsurface antigen selected from the group consisting of: GONMB, TACSTD2(TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-α,folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1,mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b,CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16,Tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin, fibronectinextra-domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin,endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2,FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7,FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15,CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38,CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138,cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA1,EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3,EphB4, EphB6, an integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA,c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB,TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope on a cell surface antigen(s) derivedfrom, or determined to be expressed on, a specific subject's cancer(tumor) such as a neoantigen. In some embodiments, the contacted cancercell is a mammalian cell. In further embodiments, the contacted cancercell is a human cell. In additional embodiments, the contacted cancercell is a primary cell or a cell from a cell line obtained/derived froma cancer selected from the group consisting of: lung cancer (e.g.,non-small cell), pancreatic cancer, breast cancer, ovarian cancer,prostate cancer, head and neck cancer, gastric cancer, gastrointestinalcancer, colorectal cancer, esophageal cancer, cervical cancer, livercancer, kidney cancer, biliary duct cancer, gallbladder cancer, bladdercancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervoussystem cancer, melanoma, myeloma, a leukemia and a lymphoma. In someembodiments, the contacted cancer cell is a primary cell or a cell froma cell line obtained/derived from a cancer selected from the groupconsisting of: colorectal cancer, breast cancer, gastric cancer (e.g.,stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g.,non-small cell lung cancer and/or adenocarcinoma), head and neck cancer,ovarian cancer, gallbladder cancer, and basal cell cancer. In particularembodiments, the contacted cancer cell is a primary cell or a cell froma cell line obtained/derived from colorectal cancer. In someembodiments, the method is performed in vivo. In some embodiments, themethod is performed in vitro. In other embodiments, the targetingvehicle is a liposome that comprises a targeting moiety that hasspecific affinity for an epitope on one of the above-listed cell surfaceantigens. In some embodiments, the liposome is pegylated. In otherembodiments, the liposome that is unpegylated. In some embodiments, theliposome comprises a αPTHF composition containing 4, 5, 2-10, 4-6, ormore than 5, glutamyl groups. In some embodiments, the liposomecomprises tetraglutamated alpha tetrahydrofolate. In some embodiments,the liposome comprises pentaglutamated alpha tetrahydrofolate. In otherembodiments, the liposome comprises hexaglutamated alphatetrahydrofolate. In some embodiments, the liposome comprises a αPTHFselected from: (a) polyglutamated 5-formyl-THF (e.g., polyglutamated[6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF (e.g.,polyglutamated [6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF(e.g., polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF); (e)polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, the liposome comprises polyglutamated 5,10-methylene-THF.In further embodiments, the liposome comprises polyglutamated[6R]-5,10-methylene-THF. In other embodiments, the liposome comprisespolyglutamated [6R,S]-5,10-methylene-THF. In some embodiments, theliposome comprises polyglutamated 5-methyl-THF. In further embodiments,the liposome comprises [6S]-5-methyl-THF. In other embodiments, theliposome comprises [6R,S]-5-methyl-THF. In some embodiments, theliposome comprises polyglutamated 5-formyl-THF. In further embodiments,the liposome comprises polyglutamated [6S]-5-formyl-THF. In otherembodiments, the liposome comprises polyglutamated [6R,S]-5-formyl-THF.

In additional embodiments, the disclosure provides a method for treatinga hyperproliferative disorder that comprises administering an effectiveamount of a delivery vehicle (e.g., antibody or liposome) comprisingpolyglutamated alpha tetrahydrofolate (e.g., a αPTHF disclosed herein)to a subject having or at risk of having a hyperproliferative disorder.In some embodiments, the delivery vehicle is an antibody (e.g., afull-length IgG antibody, a bispecific antibody, or a scFv). In someembodiments, the delivery vehicle is a liposome (e.g., an Lp-αPTHF suchas, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF)). Insome embodiments, the administered delivery vehicle is pegylated. Insome embodiments, the administered delivery vehicle is not pegylated. Inadditional embodiments, the administered delivery vehicle comprises atargeting moiety that has specific affinity for an epitope of an antigenon the surface of the hyperproliferative cell. In additionalembodiments, the delivery vehicle comprises a targeting moiety that hasspecific affinity for an epitope of a cell surface antigen selected fromthe group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, afolate receptor (e.g., folate receptor-α, folate receptor-β or folatereceptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, Nectin 4,ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9(Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, Tissue factor, LIV-1(ZIP6), CGEN-15027, P cadherin, fibronectin extra-domain B (ED-B),VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor,HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6,IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10,SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20,CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56,CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R,IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA1, EphA2, EphA3,EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, anintegrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242 antigen, Apo2,PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1,VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK,ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK. In someembodiments, the delivery vehicle comprises a targeting moiety that hasspecific affinity for an epitope on a cell surface antigen(s) derivedfrom, or determined to be expressed on, a specific subject's cancer(tumor) such as a neoantigen. In some embodiments, the targeting moietyis an antibody or an antigen binding antibody fragment. In someembodiments, the administered delivery vehicle does not comprise atargeting moiety that has a specific affinity for an epitope of a cellsurface antigen of the hyperproliferative cell. In some embodiments, theadministered delivery vehicle comprises αPTHF containing 4, 5, 2-10,4-6, or more than 5, glutamyl groups. In some embodiments, theadministered delivery vehicle comprises tetraglutamated alphatetrahydrofolate. In some embodiments, the administered delivery vehiclecomprises pentaglutamated alpha tetrahydrofolate. In other embodiments,the administered delivery vehicle comprises hexaglutamated alphatetrahydrofolate. In some embodiments, the administered delivery vehiclecomprises a αPTHF selected from: (a) polyglutamated 5-formyl-THF (e.g.,polyglutamated [6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF(e.g., polyglutamated [6R]-10-formyl-THF); (c) polyglutamated5,10-methenyl-THF (e.g., polyglutamated [6R]-5,10-methenyl-THF); (d)polyglutamated 5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF);(e) polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, the delivery vehicle comprises polyglutamated5,10-methylene-THF. In further embodiments, the delivery vehiclecomprises polyglutamated [6R]-5,10-methylene-THF. In other embodiments,the delivery vehicle comprises polyglutamated [6R,S]-5,10-methylene-THF.In some embodiments, the delivery vehicle comprises polyglutamated5-methyl-THF. In further embodiments, the delivery vehicle comprises[6S]-5-methyl-THF. In other embodiments, the delivery vehicle comprises[6R,S]-5-methyl-THF. In some embodiments, the delivery vehicle comprisespolyglutamated 5-formyl-THF. In further embodiments, the deliveryvehicle comprises polyglutamated [6S]-5-formyl-THF. In otherembodiments, the delivery vehicle comprises polyglutamated[6R,S]-5-formyl-THF. In some embodiments, the hyperproliferativedisorder is cancer. In some embodiments, the hyperproliferative disorderis an autoimmune disease (e.g., inflammation and rheumatoid arthritis).In some embodiments, the hyperproliferative disorder is a benign ormalignant tumor; leukemia, hematological, or lymphoid malignancy. Inother embodiments, the hyperproliferative disorder selected from thegroup consisting of a: neuronal, glial, astrocytal, hypothalamic,glandular, macrophagal, epithelial, stromal, blastocoelic, inflammatory,angiogenic and immunologic disorder, including an autoimmune disease.

In additional embodiments, the disclosure provides a method for treatinga hyperproliferative disorder that comprises administering an effectiveamount of a liposome comprising polyglutamated alpha tetrahydrofolate(e.g., an Lp-αPTHF such as, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF,TLp-αPTHF, or TPLp-αPTHF) to a subject having or at risk of having ahyperproliferative disorder. In some embodiments, the liposome ispegylated. In some embodiments, the liposome is not pegylated. Inadditional embodiments, the liposome comprises a targeting moiety thathas a specific affinity for an epitope of an antigen on the surface ofthe hyperproliferative cell. In additional embodiments, the liposomecomprises a targeting moiety that has a specific affinity for an epitopeof a cell surface antigen selected from the group consisting of: GONMB,TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin,fibronectin extra-domain B (ED-B), VEGFR2 (CD309), tenascin, collagenIV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, the liposomecomprises a targeting moiety that has a specific affinity for an epitopeon cell surface antigen(s) derived from, or determined to be expressedon, a specific subject's cancer (tumor) such as a neoantigen. In someembodiments, the targeting moiety is an antibody or an antigen bindingantibody fragment. In some embodiments, the liposome does not comprise atargeting moiety that has a specific affinity for an epitope of a cellsurface antigen of the hyperproliferative cell. In some embodiments, theliposome comprises αPTHF containing 4, 5, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, the liposome comprisestetraglutamated alpha tetrahydrofolate. In some embodiments, theliposome comprises pentaglutamated alpha tetrahydrofolate. In otherembodiments, the liposome comprises hexaglutamated alphatetrahydrofolate. In some embodiments, the liposome comprises a αPTHFselected from: (a) polyglutamated 5-formyl-THF (e.g., polyglutamated[6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF (e.g.,polyglutamated [6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF(e.g., polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF); (e)polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, the liposome comprises polyglutamated 5,10-methylene-THF.In further embodiments, the liposome comprises polyglutamated[6R]-5,10-methylene-THF. In other embodiments, the liposome comprisespolyglutamated [6R,S]-5,10-methylene-THF. In some embodiments, theliposome comprises polyglutamated 5-methyl-THF. In further embodiments,the liposome comprises [6S]-5-methyl-THF. In other embodiments, theliposome comprises [6R,S]-5-methyl-THF. In some embodiments, theliposome comprises polyglutamated 5-formyl-THF. In further embodiments,the liposome comprises polyglutamated [6S]-5-formyl-THF. In otherembodiments, the liposome comprises polyglutamated [6R,S]-5-formyl-THF.In some embodiments, the liposome comprises L polyglutamated alphatetrahydrofolate. In some embodiments, the hyperproliferative disorderis cancer. In some embodiments, the hyperproliferative disorder is anautoimmune disease (e.g., rheumatoid arthritis). In some embodiments,the hyperproliferative disorder is a benign or malignant tumor;leukemia, hematological, or lymphoid malignancy. In other embodiments,the hyperproliferative disorder is selected from the group consisting ofa: neuronal, glial, astrocytal, hypothalamic, glandular, macrophagal,epithelial, stromal, blastocoelic, inflammatory, angiogenic andimmunologic disorder, including an autoimmune disease.

Exemplary hyperproliferative disorders that can be treated according tothe disclosed methods include, but are not limited to, disordersassociated with benign, pre-malignant, and malignant cellularproliferation, including but not limited to, neoplasms and tumors (e.g.,histiocytoma, glioma, astrocytoma, osteoma), cancers (e.g., lung cancer,small cell lung cancer, gastrointestinal cancer, bowel cancer,colorectal cancer, breast carcinoma, ovarian carcinoma, prostate cancer,testicular cancer, liver cancer, kidney cancer, bladder cancer,pancreatic cancer, brain cancer, sarcoma (e.g., osteosarcoma, Kaposi'ssarcoma), and melanoma), leukemias, psoriasis, bone diseases,fibroproliferative disorders (e.g., of connective tissues), andatherosclerosis.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a deliveryvehicle (e.g., antibody or liposome) comprising polyglutamated alphatetrahydrofolate (e.g., a αPTHF disclosed herein) to a subject having orat risk of having cancer. In some embodiments, the delivery vehicle isan antibody (e.g., a full-length IgG antibody, a bispecific antibody, ora scFv). In some embodiments, the delivery vehicle is a liposome (e.g.,an Lp-αPTHF such as, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, orTPLp-αPTHF). In some embodiments, the administered delivery vehicle ispegylated. In some embodiments, the administered delivery vehicle is notpegylated. In additional embodiments, the administered delivery vehiclecomprises a targeting moiety that has a specific affinity for an epitopeof an antigen on the surface of a cancer cell. In additionalembodiments, the delivery vehicle comprises a targeting moiety that hasa specific affinity for an epitope of a cell surface antigen selectedfrom the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, afolate receptor (e.g., folate receptor-α, folate receptor-β or folatereceptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, Nectin 4,ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9(Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, Tissue factor, LIV-1(ZIP6), CGEN-15027, P cadherin, fibronectin extra-domain B (ED-B),VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor,HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6,IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10,SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20,CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56,CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R,IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA1, EphA2, EphA3,EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, anintegrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242 antigen, Apo2,PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1,VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK,ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK. In someembodiments, the delivery vehicle comprises a targeting moiety that hasspecific affinity for an epitope on a cell surface antigen(s) derivedfrom, or determined to be expressed on, a specific subject's cancer(tumor) such as a neoantigen. In some embodiments, the targeting moietyis an antibody or an antigen binding antibody fragment. In someembodiments, the administered delivery vehicle comprises αPTHFcontaining 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the administered delivery vehicle comprises tetraglutamatedalpha tetrahydrofolate. In some embodiments, the administered deliveryvehicle comprises pentaglutamated alpha tetrahydrofolate. In otherembodiments, the administered delivery vehicle comprises hexaglutamatedalpha tetrahydrofolate. In some embodiments, the administered deliveryvehicle comprises a αPTHF selected from: (a) polyglutamated 5-formyl-THF(e.g., polyglutamated [6S]-5-formyl-THF); (b) polyglutamated10-formyl-THF (e.g., polyglutamated [6R]-10-formyl-THF); (c)polyglutamated 5,10-methenyl-THF (e.g., polyglutamated[6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF (e.g.,polyglutamated [6S]-5-methyl-THF); (e) polyglutamated TetrahydrofolateTHF (e.g., polyglutamated [6S]-Tetrahydrofolate THF); (f) polyglutamated5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and(g) polyglutamated 5-formimino-THF (e.g., polyglutamated[6S]-5-formimino-THF). In some embodiments, the administered deliveryvehicle comprises polyglutamated 5,10-methylene-THF. In furtherembodiments, the administered delivery vehicle comprises polyglutamated[6R]-5,10-methylene-THF. In other embodiments, the administered deliveryvehicle comprises polyglutamated [6R,S]-5,10-methylene-THF. In someembodiments, the administered delivery vehicle comprises polyglutamated5-methyl-THF. In further embodiments, the administered delivery vehiclecomprises [6S]-5-methyl-THF. In other embodiments, the administereddelivery vehicle comprises [6R,S]-5-methyl-THF. In some embodiments, theadministered delivery vehicle comprises polyglutamated 5-formyl-THF. Infurther embodiments, the administered delivery vehicle comprisespolyglutamated [6S]-5-formyl-THF. In other embodiments, the administereddelivery vehicle comprises polyglutamated [6R,S]-5-formyl-THF. In someembodiments, the cancer is selected from the group consisting of: lung(e.g., non-small lung cancer), pancreatic, breast cancer, ovarian, lung,prostate, head and neck, gastric, gastrointestinal, colon, esophageal,cervical, kidney, biliary duct, gallbladder, and a hematologicmalignancy (e.g., a leukemia or lymphoma).

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomecomprising polyglutamated alpha tetrahydrofolate (e.g., an Lp-αPTHF suchas, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF) to asubject having or at risk of having cancer. In some embodiments, theliposome is pegylated. In some embodiments, the liposome is notpegylated. In additional embodiments, the liposome comprises a targetingmoiety that has a specific affinity for an epitope of an antigen on thesurface of a cancer cell. In additional embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeof a cell surface antigen selected from the group consisting of: GONMB,TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin,fibronectin extra-domain B (ED-B), VEGFR2 (CD309), tenascin, collagenIV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeon a cell surface antigen(s) derived from, or determined to be expressedon, a specific subject's cancer (tumor) such as a neoantigen. In someembodiments, the targeting moiety is an antibody or an antigen bindingantibody fragment. In some embodiments, the liposome comprises αPTHFcontaining 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the liposome comprises tetraglutamated alphatetrahydrofolate. In some embodiments, the liposome comprisespentaglutamated alpha tetrahydrofolate. In other embodiments, theliposome comprises hexaglutamated alpha tetrahydrofolate. In someembodiments, the administered liposome comprises a αPTHF selected from:(a) polyglutamated 5-formyl-THF (e.g., polyglutamated[6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF (e.g.,polyglutamated [6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF(e.g., polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF); (e)polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, the administered liposome comprises polyglutamated5,10-methylene-THF. In further embodiments, the administered liposomecomprises polyglutamated [6R]-5,10-methylene-THF. In other embodiments,the administered liposome comprises polyglutamated[6R,S]-5,10-methylene-THF. In some embodiments, the administeredliposome comprises polyglutamated 5-methyl-THF. In further embodiments,the administered liposome comprises [6S]-5-methyl-THF. In otherembodiments, the administered liposome comprises [6R,S]-5-methyl-THF. Insome embodiments, the administered liposome comprises polyglutamated5-formyl-THF. In further embodiments, the administered liposomecomprises polyglutamated [6S]-5-formyl-THF. In other embodiments, theadministered liposome comprises polyglutamated [6R,S]-5-formyl-THF. Insome embodiments, the cancer is selected from the group consisting of:lung (e.g., non-small lung cancer), pancreatic, breast cancer, ovarian,lung, prostate, head and neck, gastric, gastrointestinal, colon,esophageal, cervical, kidney, biliary duct, gallbladder, and ahematologic malignancy (e.g., a leukemia or lymphoma).

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering to a subject having or at risk ofhaving cancer, an effective amount of a liposomal composition containinga liposome that comprises polyglutamated alpha tetrahydrofolate (e.g., aαPTHF disclosed herein) and a targeting moiety that has a specificaffinity for an epitope of an antigen on the surface of the cancer. Insome embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of a cell surface antigen selected fromthe group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, afolate receptor (e.g., folate receptor-α, folate receptor-β or folatereceptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, Nectin 4,ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9(Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, Tissue factor, LIV-1(ZIP6), CGEN-15027, P cadherin, fibronectin extra-domain B (ED-B),VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor,HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6,IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10,SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20,CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56,CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R,IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA1, EphA2, EphA3,EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, anintegrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242 antigen, Apo2,PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1,VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK,ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK. In someembodiments, the liposome comprises a targeting moiety that has specificaffinity for an epitope on a cell surface antigen(s) derived from, ordetermined to be expressed on, a specific subject's cancer (tumor) suchas a neoantigen. In some embodiments, the administered liposomalcomposition comprises pegylated liposomes (e.g., TPLp-αPTHF). In someembodiments, the administered liposomal composition comprises liposomesthat are not pegylated. In some embodiments, liposomes of theadministered liposomal composition comprises αPTHF containing 4, 5,2-10, 4-6, or more than 5, glutamyl groups. In some embodiments,liposomes of the administered liposomal composition comprisetetraglutamated alpha tetrahydrofolate. In some embodiments, liposomesof the administered liposomal composition comprise pentaglutamated alphatetrahydrofolate. In other embodiments, liposomes of the administeredliposomal composition comprises hexaglutamated alpha tetrahydrofolate.In some embodiments, administered liposomes comprise a αPTHF selectedfrom: (a) polyglutamated 5-formyl-THF (e.g., polyglutamated[6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF (e.g.,polyglutamated [6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF(e.g., polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF); (e)polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, t administered liposomes comprise polyglutamateadministered liposomes comprise d 5,10-methylene-THF. In furtherembodiments, administered liposomes comprise polyglutamated[6R]-5,10-methylene-THF. In other embodiments, administered liposomescomprise polyglutamated [6R,S]-5,10-methylene-THF. In some embodiments,administered liposome comprises polyglutamated 5-methyl-THF. In furtherembodiments, administered liposome comprises [6S]-5-methyl-THF. In otherembodiments, administered liposome comprises [6R,S]-5-methyl-THF. Insome embodiments, administered liposome comprises polyglutamated5-formyl-THF. In further embodiments, administered liposome comprisespolyglutamated [6S]-5-formyl-THF. In other embodiments, administeredliposome comprises polyglutamated [6R,S]-5-formyl-THF. In someembodiments, the liposomal composition is administered to treat a cancerselected from the group consisting of: lung cancer, pancreatic, breastcancer, ovarian cancer, lung cancer, prostate cancer, head and neckcancer, gastric cancer, gastrointestinal cancer, colon cancer,esophageal cancer, cervical cancer, kidney cancer, biliary duct cancer,gallbladder cancer, and a hematologic malignancy.

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of a tumor specific antigen (TSA) ortumor associated antigen (TAA). In some embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeof an antigen selected from the group consisting of: a tumordifferentiation antigen (e.g., MART1/MelanA, gp100 (Pmel 17),tyrosinase, TRP1, and TRP2), a tumor-specific multilineage antigen(e.g., MAGE1, MAGE3, BAGE, GAGE1, GAGE2, and p15), an overexpressedembryonic antigen (e.g., carcinoembryonic antigen (CEA)), anoverexpressed oncogene or mutated tumor-suppressor gene product (e.g.,p53, Ras, and HER2/neu), a unique tumor antigen resulting fromchromosomal translocations (e.g., BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, andMYL-RAR), a viral antigen (e.g., Epstein Barr virus antigen EBVA, humanpapillomavirus (HPV) antigen E6 or E7), GP 100), prostatic acidphosphatase (PAP), prostate-specific antigen (PSA), PTGER4, ITGA4, CD37,CD52, CD62L (L-selectin), CXCR4, CD69, EVI2B (CD361), SLC39A8, MICB,LRRC70, CLELC2B, HMHA1, LST1, and CMTM6 (CKLFSF6).

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of a hematologic tumor antigen. Infurther embodiments, the targeting moiety has specific affinity for anepitope of a hematologic tumor antigen selected from the groupconsisting of: CD19, CD20, CD22, CD30, CD138, CD33, CD38, CD123, CS1,ROR1, Lewis^(Y), Ig kappa light chain, TCR, BCMA, TACI, BAFFR (CD268),CALLA, and a NKG2DL ligand). In some embodiments, the liposome comprisesa targeting moiety that has specific affinity for an epitope of a B-celllymphoma-specific idiotype immunoglobulin, or a B-cell differentiationantigen (e.g., CD19, CD20, and CD37). In some embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeof an antigen on a multiple myeloma cell (e.g., CS-1, CD38, CD138, MUC1,HM1.24, CYP1B1, SP17, PRAME, Wilms' tumor 1 (WT1), and heat shockprotein gp96) or an antigen on myeloid cells (e.g., TSLPR and IL-7R).

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of a solid tumor antigen. In furtherembodiments, the targeting moiety has specific affinity for an epitopeof a hematologic tumor antigen selected from the group consisting of:disialoganglioside (GD2), o-acetyl GD2, EGFRvIII, ErbB2, VEGFR2, FAP,mesothelin, IL13Ra2 (glioma), cMET, PSMA, L1CAM, CEA, and EGFR.

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of an antigen selected from the groupconsisting of: CD137, PDL1, CTLA4, CD47, KIR, TNFRSF10B (DR5), TIM3,PD1, cMet, Glycolipid F77, EGFRvIII, HLAA2 (NY-ESO-1), LAG3, CD134(OX40), HVEM, BTLA, TNFRSF25 (DR3), CD133, MAGE A3, PSCA, MUC1, CD44v6,CD44v6/7, CD44v7/8, IL11Ra, ephA2, CAIX, MNCAIX, CSPG4, MUC16, EPCAM(EGP2), TAG72, EGP40, ErbB receptor family, ErbB2 (HER2), ErbB3/4,RAGE1, GD3, FAR, Lewis^(Y), NCAM, HLAA1/MAGE1, MAGEA1, MAGEA3, MAGE-A4,B7H3, WT1, MelanA (MART1), HPV E6, HPV E7, thyroglobulin, tyrosinase,PSA, CLL1GD3, Tn Ag, FLT3, KIT, PRSS21, CD24, PDGFR-beta, SSEA4,prostase, PAP, ELF2M, ephB2, IGF1, IGFII, IGFI receptor, LMP2, gp100,bcr-ab1, Fucosyl GM1, sLe, GM3, TGS5, folate receptor beta, TEM1(CD248), TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD7a, HLE, CD179a,ALK, Plysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3,GPR20, LY6K, OR51E2, TARP, LAGE1a, legumain, E7, ETV6-AML, sperm protein17, XAGE1, Tie 2, MAD-CT1, MAD-CT2, Fos-related antigen 1, p53, p53mutant, prostein, survivin, telomerase, PCTA1 (Galectin 8), Ras mutant,hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETSfusion gene), NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC,TRP2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP4, SSX2, reversetranscriptase, RU1, RU2, intestinal carboxyl esterase, neutrophilelastase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF,CLEC12A, BST2, EMR2, LY75, GPC3, FCRLS, IGLL1, TSP-180, MAGE4, MAGE5,MAGE6, VEGFR1, IGF1R, hepatocyte growth factor receptor, p185ErbB2,p180ErbB-3, nm-23H1, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,beta-Catenin, CDK4, Mum1, p15, p16, 43-9F, 5T4, 791Tgp72, β-humanchorionic gonadotropin, BCA225, BTAA, CA125, CA15-3, CA 27.29 (BCAA),CA195, CA242, CA-50, CAM43, CD68, CO-029, FGF5, G250, HTgp-175, M344,MA50, MG7-Ag, MOV18, NB/70K, NY-CO1, RCAS1, SDCCAG16, M2BP, TAAL6, TLP,and TPS, glioma-associated antigen, alpha-fetoprotein (AFP), p26fragment of AFP, lectin-reactive AFP, and TLR4.

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of an antigen selected from the groupconsisting of: PDGFRA, VEGFR1, VEGFR3, neuropilin 1 (NRP1), neuropilin 2(NRP2), betacellulin, PLGF, RET (rearranged during transfection), TIE1,TIE2 (TEK), CA125, CD3, CD4, CD7, CD10, CD13, CD25 CD32, CD32b, CD44(e.g., CD44v6), CD47, CD49e (integrin alpha 5), CD54 (ICAM), CD55, CD64,CD74, CD80, CD90, CD200, CD147, CD166, CD200, ESA, SHH, DHH, IHH,patched 1 (PTCH1), smoothened (SMO), WNT1, WNT2B, WNT3A, WNT4, WNT4A,WNT5A, WNT5B, WNT7B, WNT8A, WNT10A, WNT10B, WNT16B, LKP5, LRP5, LRP6,FZD1, FZD2, FZD4, FZD5, FZD6, FZD7, FZD8, Notch, Notch1, Notch3, Notch4,DLL4, Jagged, Jagged1, Jagged2, Jagged3, TNFRSF1A (TNFR1, p55, p60),TNFRSF1B (TNFR2), TNFRSF6 (Fas, CD95), TNFRSF6B (DcR3), TNFRSF7 (CD27),TNFSF9 (41BB Ligand), TNFRSF8 (CD30), TNFRSF10A (TRAILR1, DR4),TNFRSF11A (RANK), TNFRSF12 (TWEAKR), TNFRSF19L (KELT), TNFRSF19 (TROY),TNFRSF21 (DR6), ILIRI, 1L1R2, IL2R, IL5R, IL6R, 1L8R, IL10R, IL12R,IL13R, IL15R, IL18R, IL19R, IL21R, IL23R, XAG1, XAG3, REGIV, FGFR1,FGFR2, FGFR3, ALK, ALK1, ALK7, ALCAM, Axl, TGFb, TGFb2, TGFb3, TGFBR1,IGFIIR, BMPRI, N-cadherin, E-cadherin, VE-cadherin, ganglioside GM2,ganglioside GD3, PSGR, DCC, CDCP1, CXCR2, CXCR7, CCR3, CCR4, CCR5, CCR7,CCR10, Claudin1, Claudin2, Claudin3, Claudin4, TMEFF2, neuregulin, MCSF,CSF, CSFR (fms), GCSF, GCSFR, BCAM, BRCA1, BRCA2, HLA-DR, ABCC3, ABCB5,HM 1.24, LFA1, LYNX, S100A8, S100A9, SCF, Von Willebrand factor, LewisY6 receptor, CA G250 (CA9), CRYPTO, VLA5, HLADR, MUC18, mucin CanAg,EGFL7, integrin avb3, integrin α5β activin Bl alpha, leukotriene B4receptor (LTB4R), neurotensin NT receptor (NTR), 5T4 oncofetal antigen,Tenascin C, MMP, MMP2, MMP7, MMP9, MMP12, MMP14, MMP26, cathepsin G,SULF1, SULF2, MET, CA9, TM4SF1, syndecan (SDC1), Ephrin B4, TEM1,TGFbeta 1, and TGFBRII.

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of an antigen associated with adisorder of the immune system (e.g., an autoimmune disorder and aninflammatory disorder), or is associated with regulating an immuneresponse. In some embodiments, the targeting moiety has specificaffinity for an epitope of a cell surface antigen expressed on thesurface of a macrophage (expressing CD44).

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of an immunoinhibitory target. Inanother embodiment, the AD is an epitope of an immunoinhibitory targetselected from the group consisting of: IL1Ra, IL6R, CD26L, CD28, CD80,Fcgamma RIM In another embodiment, the AD in the Adapter is an epitopeof an immunostimulatory target selected from: CD25, CD28, CTLA4, PD1,B7H1 (PDL1), B7H4 TGFbeta, TNFRSF4 (OX40), TNFRSF5 (CD40), TNFRSF9(41BB, CD137), TNFRSF14 (HVEM), TNFRSF25 (DR3), and TNFRSF18 (GITR).

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of an antigen selected from the groupconsisting of: IL1Rb, C3AR, C5AR, CXCR1, CXCR2, CCR1, CCR3, CCR7, CCR8,CCR9, CCR10, ChemR23, MPL, GP130, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8,TLR9, TREM1, TREM2, CD49a (integrin alpha 1), integrin a5b3, alpha4integrin subunit, A4B7 integrin, cathepsin G, TNFRSF3 (LTBR), TNFRSF6(Fas, CD95), TNFRSF6B (DcR3), TNFRSF8 (CD30), TNFRSF11A (RANK), TNFRSF16(NGFR), TNFRSF19L (RELT), TNFRSF19 (TROY), TNFRSF21 (DR6), CD14, CD23,CD36, CD36L, CD39, CD91, CD153, CD164, CD200, CD200R, B71 (CD80), B72(CD86), B7h, B7DC (PDL2), ICOS, ICOSL, MHC, CD, B7H2, B7H3, B7x, SLAM,KIM1, SLAMF2, SLAMF3, SLAMF4, SLAMF5, SLAMF6, SLAMF7, TNFRSF1A (TNFR1,p55, p60), TNFRSF1B (TNFR2), TNFRSF7 (CD27), TNFRSF12 (TWEAKR), TNFRSF5(CD40), IL1R, IL2R, IL4Ra, IL5R, IL6RIL15R, IL17R, IL17Rb, IL17RC,IL22RA, IL23R, TSLPR, B7RP1, cKit, GMCSF, GMCSFR, CD2, CD4, CD11a, CD18,CD30, CD40, CD86, CXCR3, CCR2, CCR4, CCR5, CCR8, RhD, IgE, and Rh.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomalcomposition to a subject having or at risk of having a cancer thatexpresses folate receptor on its cell surface, wherein the liposomalcomposition comprises liposomes that comprise (a) polyglutamated alphatetrahydrofolate (e.g., a αPTHF disclosed herein)) and (b) a targetingmoiety that has specific binding affinity for a folate receptor. In someembodiments, the targeting moiety has specific binding affinity forfolate receptor alpha (FR-α), folate receptor beta (FR-β), and/or folatereceptor delta (FR-δ). In some embodiments, the targeting moiety has aspecific binding affinity for folate receptor alpha (FR-α), folatereceptor beta (FR-β), and/or folate receptor delta (FR-δ). In someembodiments, the targeting moiety has a specific binding affinity forfolate receptor alpha (FR-α) and folate receptor beta (FR-β). In someembodiments, the administered liposomal composition comprises pegylatedliposomes (e.g., TPLp-αPTHF). In some embodiments, the administeredliposomal composition comprises liposomes that are not pegylated. Insome embodiments, liposomes of the administered liposomal compositioncomprises αPTHF containing 4, 5, 2-10, 4-6, or more than 5, glutamylgroups. In some embodiments, a liposome of the liposomal compositioncomprises tetraglutamated alpha tetrahydrofolate. In some embodiments, aliposome of the liposomal composition comprises pentaglutamated alphatetrahydrofolate. In some embodiments, a liposome of the liposomalcomposition comprises alpha hexaglutamated tetrahydrofolate. In someembodiments, administered liposomes comprise a αPTHF selected from: (a)polyglutamated 5-formyl-THF (e.g., polyglutamated [6S]-5-formyl-THF);(b) polyglutamated 10-formyl-THF (e.g., polyglutamated[6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF (e.g.,polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF(e.g., polyglutamated [6S]-5-methyl-THF); (e) polyglutamatedTetrahydrofolate THF (e.g., polyglutamated [6S]-Tetrahydrofolate THF);(f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated[6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g.,polyglutamated [6S]-5-formimino-THF). In some embodiments, administeredliposomes comprise polyglutamated 5,10-methylene-THF. In furtherembodiments, administered liposomes comprise polyglutamated[6R]-5,10-methylene-THF. In other embodiments, administered liposomescomprise polyglutamated [6R,S]-5,10-methylene-THF. In some embodiments,administered liposomes comprise polyglutamated 5-methyl-THF. In furtherembodiments, administered liposomes comprise [6S]-5-methyl-THF. In otherembodiments, administered liposomes comprise [6R,S]-5-methyl-THF. Insome embodiments, administered liposomes comprise polyglutamated5-formyl-THF. In further embodiments, administered liposomes comprisepolyglutamated [6S]-5-formyl-THF. In other embodiments, administeredliposomes comprise polyglutamated [6R,S]-5-formyl-THF.

In some embodiments, a liposome of the liposomal composition comprises aαPTHF containing 2, 3, 4, 5, or more than 5, glutamyl groups. In someembodiments, liposomes of the administered liposomal compositioncomprise tetraglutamated alpha tetrahydrofolate. In some embodiments,liposomes of the administered liposomal composition comprisepentaglutamated alpha tetrahydrofolate. In some embodiments, liposomesof the administered liposomal composition comprises hexaglutamated alphatetrahydrofolate. In some embodiments, the liposomal composition isadministered to treat an epithelial tissue malignancy. In someembodiments, the liposomal composition is administered to treat a cancerselected from the group consisting of: lung cancer, pancreatic, breastcancer, ovarian cancer, lung cancer, prostate cancer, head and neckcancer, gastric cancer, gastrointestinal cancer, colon cancer,esophageal cancer, cervical cancer, kidney cancer, biliary duct cancer,gallbladder cancer, and a hematologic malignancy.

In some embodiments, the disclosure provides a method for treating lungcancer (e.g., non-small lung cancer) that comprises administering aneffective amount of a delivery vehicle (e.g., an antibody or liposome)comprising polyglutamated alpha tetrahydrofolate (e.g., a αPTHFdisclosed herein) to a subject having or at risk of having lung cancer.In particular embodiments, the, the cancer is non-small cell lungcancer. In some embodiments, the delivery vehicle is an antibody (e.g.,a full-length IgG antibody, a bispecific antibody, or a scFv). In someembodiments, the delivery vehicle is a liposome (e.g., an Lp-αPTHF suchas, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF)). Insome embodiments, the administered delivery vehicle is pegylated. Insome embodiments, the administered delivery vehicle is not pegylated. Inadditional embodiments, the delivery vehicle comprises a targetingmoiety on its surface that has specific affinity for an epitope of anantigen on the surface of a lung cancer (e.g., non-small cell lungcancer) cell. In further embodiments, the delivery vehicle comprises atargeting moiety that has specific affinity for an epitope of an antigenselected from the group consisting of Mucin 1, Nectin 4, NaPi2b, CD56,EGFR, and SC-16. In some embodiments, the targeting moiety is anantibody or a fragment of an antibody. In additional embodiments, thedelivery vehicle is a liposome, and the liposome comprises a targetingmoiety that has specific affinity for an epitope of an antigen selectedfrom the group consisting of Mucin 1, Nectin 4, NaPi2b, CD56, EGFR, andSC-16. In further embodiments, the delivery vehicle is a pegylatedliposome that comprises a targeting moiety that has specific affinityfor an epitope of an antigen selected from consisting of Mucin 1, Nectin4, NaPi2b, CD56, EGFR, and SC-16. In some embodiments, the administereddelivery vehicle comprises αPTHF containing 4, 5, 2-10, 4-6, or morethan 5, glutamyl groups. In some embodiments, the administered deliveryvehicle comprises a αPTHF selected from: (a) polyglutamated 5-formyl-THF(e.g., polyglutamated [6S]-5-formyl-THF); (b) polyglutamated10-formyl-THF (e.g., polyglutamated [6R]-10-formyl-THF); (c)polyglutamated 5,10-methenyl-THF (e.g., polyglutamated[6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF (e.g.,polyglutamated [6S]-5-methyl-THF); (e) polyglutamated TetrahydrofolateTHF (e.g., polyglutamated [6S]-Tetrahydrofolate THF); (f) polyglutamated5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and(g) polyglutamated 5-formimino-THF (e.g., polyglutamated[6S]-5-formimino-THF). In some embodiments, the administered deliveryvehicle comprises polyglutamated 5,10-methylene-THF. In furtherembodiments, the administered delivery vehicle comprises polyglutamated[6R]-5,10-methylene-THF. In other embodiments, the administered deliveryvehicle comprises polyglutamated [6R,S]-5,10-methylene-THF. In someembodiments, the administered delivery vehicle comprises polyglutamated5-methyl-THF. In further embodiments, the administered delivery vehiclecomprises [6S]-5-methyl-THF. In other embodiments, the administereddelivery vehicle comprises [6R,S]-5-methyl-THF. In some embodiments, theadministered delivery vehicle comprises polyglutamated 5-formyl-THF. Infurther embodiments, the administered delivery vehicle comprisespolyglutamated [6S]-5-formyl-THF. In other embodiments, the administereddelivery vehicle comprises polyglutamated [6R,S]-5-formyl-THF. In someembodiments, the administered delivery vehicle comprises tetraglutamatedalpha tetrahydrofolate. In some embodiments, the administered deliveryvehicle comprises pentaglutamated alpha tetrahydrofolate. In otherembodiments, the administered delivery vehicle comprises hexaglutamatedalpha tetrahydrofolate.

In some embodiments, the disclosure provides a method for treatingpancreatic cancer that comprises administering an effective amount of adelivery vehicle (e.g., an antibody (ADC) or liposome) comprisingpolyglutamated alpha tetrahydrofolate (e.g., a αPTHF disclosed herein)to a subject having or at risk of having pancreatic cancer. In someembodiments, the delivery vehicle is an antibody (e.g., a full-lengthIgG antibody, a bispecific antibody, or a scFv). In some embodiments,the delivery vehicle is a liposome (e.g., an Lp-αPTHF such as,PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF)). In someembodiments, the administered delivery vehicle is pegylated. In someembodiments, the administered delivery vehicle is not pegylated. Inadditional embodiments, the delivery vehicle comprises a targetingmoiety on its surface that has specific affinity for an epitope of anantigen on the surface of a pancreatic cancer cell. In furtherembodiments, the delivery vehicle comprises a targeting moiety that hasspecific affinity for an epitope of an antigen selected from the groupconsisting of TACSTD2 (TROP2), Mucin 1, mesothelin, Guanylyl cyclase C(GCC), SLC44A4, and Nectin 4. In further embodiments, the deliveryvehicle is a liposome, and the liposome comprises a targeting moiety hasspecific affinity for an epitope of an antigen selected from the groupconsisting of TACSTD2 (TROP2), Mucin 1, Mesothelin, Guanylyl cyclase C(GCC), SLC44A4, and Nectin 4. In some embodiments, the administereddelivery vehicle comprises αPTHF containing 4, 5, 2-10, 4-6, or morethan 5, glutamyl groups. In some embodiments, the administered deliveryvehicle comprises tetraglutamated alpha tetrahydrofolate. In someembodiments, the administered delivery vehicle comprises pentaglutamatedalpha tetrahydrofolate. In other embodiments, the administered deliveryvehicle comprises hexaglutamated alpha tetrahydrofolate. In someembodiments, the administered delivery vehicle comprises a αPTHFselected from: (a) polyglutamated 5-formyl-THF (e.g., polyglutamated[6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF (e.g.,polyglutamated [6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF(e.g., polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF); (e)polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, the administered delivery vehicle comprises polyglutamated5,10-methylene-THF. In further embodiments, the administered deliveryvehicle comprises polyglutamated [6R]-5,10-methylene-THF. In otherembodiments, the administered delivery vehicle comprises polyglutamated[6R,S]-5,10-methylene-THF. In some embodiments, the administereddelivery vehicle comprises polyglutamated 5-methyl-THF. In furtherembodiments, the administered delivery vehicle comprises[6S]-5-methyl-THF. In other embodiments, the administered deliveryvehicle comprises [6R,S]-5-methyl-THF. In some embodiments, theadministered delivery vehicle comprises polyglutamated 5-formyl-THF. Infurther embodiments, the administered delivery vehicle comprisespolyglutamated [6S]-5-formyl-THF. In other embodiments, the administereddelivery vehicle comprises polyglutamated [6R,S]-5-formyl-THF.

In additional embodiments, the disclosure provides a method for treatingbreast cancer (e.g., triple negative breast cancer (estrogen receptor“,progesterone receptor”, and HER2)) that comprises administering aneffective amount of a delivery vehicle (e.g., an antibody or liposome)comprising polyglutamated alpha tetrahydrofolate (e.g., a αPTHFdisclosed herein) to a subject having or at risk of having breastcancer. In some embodiments, the administered delivery vehicle is aliposome that comprises polyglutamated alpha tetrahydrofolate. In someembodiments, the delivery vehicle is an antibody (e.g., a full-lengthIgG antibody, a bispecific antibody, or a scFv). In some embodiments,the delivery vehicle is a liposome (e.g., an Lp-αPTHF such as,PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF)). In someembodiments, the administered delivery vehicle is pegylated. In someembodiments, the administered delivery vehicle is not pegylated. Inadditional embodiments, the delivery vehicle comprises a targetingmoiety on its surface that has specific affinity for an epitope of anantigen on the surface of a breast cancer cell. In further embodiments,the delivery vehicle comprises a targeting moiety that has specificaffinity for an epitope of an antigen selected from the group consistingof: LIV-1 (ZIP6), EGFR, HER2, HER3, Mucin 1, GONMB, and Nectin 4. Insome embodiments, the targeting moiety is an antibody or a fragment ofan antibody. In additional embodiments, the delivery vehicle is aliposome, and the liposome comprises a targeting moiety that hasspecific affinity for an epitope of an antigen selected from the groupconsisting of: LIV-1 (ZIP6), EGFR, HER2, HER3, Mucin 1, GONMB, andNectin 4. In some embodiments, the administered delivery vehiclecomprises αPTHF containing 4, 5, 2-10, 4-6, or more than 5, glutamylgroups. In some embodiments, the administered delivery vehicle comprisestetraglutamated alpha tetrahydrofolate. In some embodiments, theadministered delivery vehicle comprises pentaglutamated alphatetrahydrofolate. In some embodiments, the administered delivery vehiclecomprises hexaglutamated alpha tetrahydrofolate. In some embodiments,the administered delivery vehicle comprises a αPTHF selected from: (a)polyglutamated 5-formyl-THF (e.g., polyglutamated [6S]-5-formyl-THF);(b) polyglutamated 10-formyl-THF (e.g., polyglutamated[6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF (e.g.,polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF(e.g., polyglutamated [6S]-5-methyl-THF); (e) polyglutamatedTetrahydrofolate THF (e.g., polyglutamated [6S]-Tetrahydrofolate THF);(f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated[6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g.,polyglutamated [6S]-5-formimino-THF). In some embodiments, theadministered delivery vehicle comprises polyglutamated5,10-methylene-THF. In further embodiments, the administered deliveryvehicle comprises polyglutamated [6R]-5,10-methylene-THF. In otherembodiments, the administered delivery vehicle comprises polyglutamated[6R,S]-5,10-methylene-THF. In some embodiments, the administereddelivery vehicle comprises polyglutamated 5-methyl-THF. In furtherembodiments, the administered delivery vehicle comprises[6S]-5-methyl-THF. In other embodiments, the administered deliveryvehicle comprises [6R,S]-5-methyl-THF. In some embodiments, theadministered delivery vehicle comprises polyglutamated 5-formyl-THF. Infurther embodiments, the administered delivery vehicle comprisespolyglutamated [6S]-5-formyl-THF. In other embodiments, the administereddelivery vehicle comprises polyglutamated [6R,S]-5-formyl-THF.

In some embodiments, the disclosure provides a method for treating ahematological cancer that comprises administering an effective amount ofa delivery vehicle (e.g., an antibody or liposome) comprisingpolyglutamated alpha tetrahydrofolate (e.g., a αPTHF disclosed herein)to a subject having or at risk of having a hematological cancer. In someembodiments, the delivery vehicle is an antibody (e.g., a full-lengthIgG antibody, a bispecific antibody, or a scFv). In some embodiments,the delivery vehicle is a liposome (e.g., an Lp-αPTHF such as,PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF)). In someembodiments, the administered delivery vehicle is pegylated. In someembodiments, the administered delivery vehicle is not pegylated. Inadditional embodiments, the delivery vehicle comprises a targetingmoiety on its surface that has specific affinity for an epitope of anantigen on the surface of a hematological cancer cell. In furtherembodiments, the delivery vehicle comprises a targeting moiety that hasspecific affinity for an epitope of an antigen selected from the groupconsisting of: CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33,and CD98. In further embodiments, the delivery vehicle is a liposome,and the liposome comprises a targeting moiety has specific affinity foran epitope of an antigen selected from the group consisting of: CD30,CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, and CD98. In someembodiments, the administered delivery vehicle comprises αPTHFcontaining 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the administered delivery vehicle comprises tetraglutamatedalpha tetrahydrofolate. In some embodiments, the administered deliveryvehicle comprises pentaglutamated alpha tetrahydrofolate. In otherembodiments, the administered delivery vehicle comprises hexaglutamatedalpha tetrahydrofolate. In some embodiments, the administered deliveryvehicle comprises a αPTHF selected from: (a) polyglutamated 5-formyl-THF(e.g., polyglutamated [6S]-5-formyl-THF); (b) polyglutamated10-formyl-THF (e.g., polyglutamated [6R]-10-formyl-THF); (c)polyglutamated 5,10-methenyl-THF (e.g., polyglutamated[6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF (e.g.,polyglutamated [6S]-5-methyl-THF); (e) polyglutamated TetrahydrofolateTHF (e.g., polyglutamated [6S]-Tetrahydrofolate THF); (f) polyglutamated5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and(g) polyglutamated 5-formimino-THF (e.g., polyglutamated[6S]-5-formimino-THF). In some embodiments, the administered deliveryvehicle comprises polyglutamated 5,10-methylene-THF. In furtherembodiments, the administered delivery vehicle comprises polyglutamated[6R]-5,10-methylene-THF. In other embodiments, the administered deliveryvehicle comprises polyglutamated [6R,S]-5,10-methylene-THF. In someembodiments, the administered delivery vehicle comprises polyglutamated5-methyl-THF. In further embodiments, the administered delivery vehiclecomprises [6S]-5-methyl-THF. In other embodiments, the administereddelivery vehicle comprises [6R,S]-5-methyl-THF. In some embodiments, theadministered delivery vehicle comprises polyglutamated 5-formyl-THF. Infurther embodiments, the administered delivery vehicle comprisespolyglutamated [6S]-5-formyl-THF. In other embodiments, the administereddelivery vehicle comprises polyglutamated [6R,S]-5-formyl-THF.

In some embodiments, the disclosure provides a method for treating asubject having or at risk of having a cancer that is distinguishable bythe expression of an antigen on its cell surface. Thus, in someembodiments, the disclosure provides a method for treating cancer thatcomprises administering to a subject having or at risk of having acancer, an effective amount of a delivery vehicle (e.g., an antibody orliposome) comprising a targeting moiety that has specific affinity foran epitope of a surface antigen of the cancer and polyglutamated alphatetrahydrofolate (e.g., a αPTHF disclosed herein). In some embodiments,the administered delivery vehicle is pegylated. In some embodiments, thetargeting moiety is an antibody or a fragment of an antibody. Inadditional embodiments, the delivery vehicle is a liposome. In someembodiments, the administered delivery vehicle comprises αPTHFcontaining 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the administered delivery vehicle comprises tetraglutamatedalpha tetrahydrofolate. In some embodiments, the administered deliveryvehicle comprises pentaglutamated alpha tetrahydrofolate. In otherembodiments, the administered delivery vehicle comprises hexaglutamatedalpha tetrahydrofolate. In some embodiments, the administered deliveryvehicle comprises a αPTHF selected from: (a) polyglutamated 5-formyl-THF(e.g., polyglutamated [6S]-5-formyl-THF); (b) polyglutamated10-formyl-THF (e.g., polyglutamated [6R]-10-formyl-THF); (c)polyglutamated 5,10-methenyl-THF (e.g., polyglutamated[6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF (e.g.,polyglutamated [6S]-5-methyl-THF); (e) polyglutamated TetrahydrofolateTHF (e.g., polyglutamated [6S]-Tetrahydrofolate THF); (f) polyglutamated5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and(g) polyglutamated 5-formimino-THF (e.g., polyglutamated[6S]-5-formimino-THF). In some embodiments, the administered deliveryvehicle comprises polyglutamated 5,10-methylene-THF. In furtherembodiments, the administered delivery vehicle comprises polyglutamated[6R]-5,10-methylene-THF. In other embodiments, the administered deliveryvehicle comprises polyglutamated [6R,S]-5,10-methylene-THF. In someembodiments, the administered delivery vehicle comprises polyglutamated5-methyl-THF. In further embodiments, the administered delivery vehiclecomprises [6S]-5-methyl-THF. In other embodiments, the administereddelivery vehicle comprises [6R,S]-5-methyl-THF. In some embodiments, theadministered delivery vehicle comprises polyglutamated 5-formyl-THF. Infurther embodiments, the administered delivery vehicle comprisespolyglutamated [6S]-5-formyl-THF. In other embodiments, the administereddelivery vehicle comprises polyglutamated [6R,S]-5-formyl-THF.

In some embodiments, the disclosed compositions (e.g., liposomescontaining polyglutamated alpha tetrahydrofolate) are administered tosubjects having or at risk of having a cancer, a solid tumor, and/or ametastasis that is distinguishable by the expression of a tumor specificantigen or tumor associated antigen on its cell surface. Thus, in someembodiments, the disclosure provides a method for treating cancer thatcomprises administering an effective amount of a delivery vehicle (e.g.,liposome) comprising a targeting moiety and polyglutamated alphatetrahydrofolate (e.g., a αPTHF disclosed herein) to a subject having orat risk of having a cancer, solid tumor, and/or metastasis that isdistinguishable by the expression of a tumor specific antigen or tumorassociated antigen on its cell surface cancer, and wherein the targetingmoiety has specific binding affinity for an epitope on an tumor specificantigen or tumor associated antigen. In some embodiments, theadministered delivery vehicle is a liposome. In further embodiments, theliposome is pegylated. In additional embodiments, the delivery vehiclecomprises a targeting moiety that has specific affinity for an epitopeof a cell surface antigen expressed on the surface of a cancer, a solidtumor, and/or a metastatic cell. In additional embodiments, thetargeting moiety has specific affinity for an epitope of a cell surfaceantigen selected from the group consisting of: GONMB, TACSTD2 (TROP2),CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-α, folatereceptor-β or folate receptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1,mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b,CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16,Tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin, fibronectinextra-domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin,endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2,FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7,FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15,CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38,CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138,cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA1,EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3,EphB4, EphB6, an integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA,c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB,TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the delivery vehicle comprises a targeting moietythat has specific affinity for an epitope on a cell surface antigen(s)derived from, or determined to be expressed on, a specific subject'scancer (tumor) such as a neoantigen. In some embodiments, theadministered delivery vehicle comprises αPTHF containing 4, 5, 2-10,4-6, or more than 5, glutamyl groups. In some embodiments, theadministered delivery vehicle comprises tetraglutamated alphatetrahydrofolate. In some embodiments, the administered delivery vehiclecomprises pentaglutamated alpha tetrahydrofolate. In other embodiments,the administered delivery vehicle comprises hexaglutamated alphatetrahydrofolate. In some embodiments, the administered delivery vehiclecomprises a αPTHF selected from: (a) polyglutamated 5-formyl-THF (e.g.,polyglutamated [6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF(e.g., polyglutamated [6R]-10-formyl-THF); (c) polyglutamated5,10-methenyl-THF (e.g., polyglutamated [6R]-5,10-methenyl-THF); (d)polyglutamated 5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF);(e) polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, the administered delivery vehicle comprises polyglutamated5,10-methylene-THF. In further embodiments, the administered deliveryvehicle comprises polyglutamated [6R]-5,10-methylene-THF. In otherembodiments, the administered delivery vehicle comprises polyglutamated[6R,S]-5,10-methylene-THF. In some embodiments, the administereddelivery vehicle comprises polyglutamated 5-methyl-THF. In furtherembodiments, the administered delivery vehicle comprises[6S]-5-methyl-THF. In other embodiments, the administered deliveryvehicle comprises [6R,S]-5-methyl-THF. In some embodiments, theadministered delivery vehicle comprises polyglutamated 5-formyl-THF. Infurther embodiments, the administered delivery vehicle comprisespolyglutamated [6S]-5-formyl-THF. In other embodiments, the administereddelivery vehicle comprises polyglutamated [6R,S]-5-formyl-THF.

In further embodiments, the delivery vehicle is a liposome, and theliposome comprises a targeting moiety that has specific affinity for acell surface antigen selected from the group consisting of: GONMB,TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin,fibronectin extra-domain B (ED-B), VEGFR2 (CD309), tenascin, collagenIV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeon a cell surface antigen(s) derived from, or determined to be expressedon, a specific subject's cancer (tumor) such as a neoantigen. In someembodiments, the administered delivery vehicle comprises αPTHFcontaining 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the liposome comprises a αPTHF selected from: (a)polyglutamated 5-formyl-THF (e.g., polyglutamated [6S]-5-formyl-THF);(b) polyglutamated 10-formyl-THF (e.g., polyglutamated[6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF (e.g.,polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF(e.g., polyglutamated [6S]-5-methyl-THF); (e) polyglutamatedTetrahydrofolate THF (e.g., polyglutamated [6S]-Tetrahydrofolate THF);(f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated[6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g.,polyglutamated [6S]-5-formimino-THF). In some embodiments, the liposomecomprises polyglutamated 5,10-methylene-THF. In further embodiments, theliposome comprises polyglutamated [6R]-5,10-methylene-THF. In otherembodiments, the liposome comprises polyglutamated[6R,S]-5,10-methylene-THF. In some embodiments, the liposome comprisespolyglutamated 5-methyl-THF. In further embodiments, the liposomecomprises [6S]-5-methyl-THF. In other embodiments, the liposomecomprises [6R,S]-5-methyl-THF. In some embodiments, the liposomecomprises polyglutamated 5-formyl-THF. In further embodiments, theliposome comprises polyglutamated [6S]-5-formyl-THF. In otherembodiments, the liposome comprises polyglutamated [6R,S]-5-formyl-THF.In some embodiments, the administered delivery vehicle comprisespentaglutamated alpha tetrahydrofolate. In other embodiments, theadministered delivery vehicle comprises hexaglutamated alphatetrahydrofolate. In some embodiments, the administered delivery vehiclecomprises L polyglutamated alpha tetrahydrofolate. In some embodiments,the administered delivery vehicle comprises D polyglutamated alphatetrahydrofolate. In some embodiments, the administered delivery vehiclecomprises L and D polyglutamated alpha tetrahydrofolate.

In further embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a deliveryvehicle (e.g., an antibody or liposome) comprising a targeting moiety onits surface has specific affinity for an epitope of a folate receptor,and a polyglutamated alpha tetrahydrofolate (e.g., a αPTHF disclosedherein) to a subject having or at risk of having a cancer that containscells expressing the folate receptor on their cell surface. In someembodiments, the targeting moiety is an antibody, or an antigen bindingfragment of an antibody. In further embodiments, the targeting moietyhas specific affinity for folate receptor alpha, folate receptor beta orfolate receptor delta. As disclosed herein, the folate receptor targetedpegylated liposomes containing polyglutamated alpha tetrahydrofolate areable to deliver high quantities of polyglutamated alpha tetrahydrofolateto cancer cells and particularly cancer cells that express folatereceptors, compared to normal cells (i.e., cells that unlike cancercells do not actively take up liposomes, and/or do not express folatereceptors). Any cancers that express folate receptors may be treatedaccording to the disclosed methods. It should be noted that some cancersmay express folate receptors in an early stage while the majority ofcancers may express folate receptors at late stages. In someembodiments, the administered delivery vehicle is a liposome. In furtherembodiments, the liposome is pegylated. In some embodiments, theadministered delivery vehicle comprises αPTHF containing 4, 5, 2-10,4-6, or more than 5, glutamyl groups. In some embodiments, theadministered delivery vehicle comprises tetraglutamated alphatetrahydrofolate. In some embodiments, the administered delivery vehiclecomprises pentaglutamated alpha tetrahydrofolate. In other embodiments,the administered delivery vehicle comprises hexaglutamated alphatetrahydrofolate. In some embodiments, the administered delivery vehiclecomprises a αPTHF selected from: (a) polyglutamated 5-formyl-THF (e.g.,polyglutamated [6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF(e.g., polyglutamated [6R]-10-formyl-THF); (c) polyglutamated5,10-methenyl-THF (e.g., polyglutamated [6R]-5,10-methenyl-THF); (d)polyglutamated 5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF);(e) polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, the administered delivery vehicle comprises polyglutamated5,10-methylene-THF. In further embodiments, the administered deliveryvehicle comprises polyglutamated [6R]-5,10-methylene-THF. In otherembodiments, the administered delivery vehicle comprises polyglutamated[6R,S]-5,10-methylene-THF. In some embodiments, the administereddelivery vehicle comprises polyglutamated 5-methyl-THF. In furtherembodiments, the administered delivery vehicle comprises[6S]-5-methyl-THF. In other embodiments, the administered deliveryvehicle comprises [6R,S]-5-methyl-THF. In some embodiments, theadministered delivery vehicle comprises polyglutamated 5-formyl-THF. Infurther embodiments, the administered delivery vehicle comprisespolyglutamated [6S]-5-formyl-THF. In other embodiments, the administereddelivery vehicle comprises polyglutamated [6R,S]-5-formyl-THF.

In additional embodiments, the disclosure provides a method for cancermaintenance therapy that comprises administering an effective amount ofa liposomal composition comprising liposomes that contain polyglutamatedalpha tetrahydrofolate (e.g., a αPTHF disclosed herein) to a subjectthat is undergoing or has undergone cancer therapy. In some embodiments,the administered liposomal composition is a PLp-αPTHF, NTLp-αPTHF,NTPLp-αPTHF, TLp-αPTHF or TPLp-αPTHF. In some embodiments, theadministered liposomal composition comprises pegylated liposomes (e.g.,PLp-αPTHF, NTPLp-αPTHF, or TPLp-αPTHF). In some embodiments, theadministered liposomal composition comprises a targeting moiety that hasspecific affinity for an epitope of a surface antigen of a cancer cell(e.g., TLp-αPTHF or TPLp-αPTHF). In some embodiments, the administeredliposomal composition comprises liposomes that are pegylated andcomprise a targeting moiety (e.g., TPLp-αPTHF). In some embodiments, theadministered liposomal composition comprises liposomes that comprise atargeting moiety and further comprises liposomes that do not comprise atargeting moiety. In some embodiments, the administered liposomalcomposition comprises liposomes that are pegylated and liposomes thatare not pegylated. In some embodiments, the administered deliveryvehicle comprises a αPTHF that contains 4, 5, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, a liposome of the administeredliposomal composition comprises tetraglutamated alpha tetrahydrofolate.In some embodiments, a liposome of the administered liposomalcomposition comprises pentaglutamated alpha tetrahydrofolate. In otherembodiments, a liposome of the administered liposomal compositioncomprises hexaglutamated alpha tetrahydrofolate. In some embodiments,the administered delivery vehicle comprises a αPTHF selected from: (a)polyglutamated 5-formyl-THF (e.g., polyglutamated [6S]-5-formyl-THF);(b) polyglutamated 10-formyl-THF (e.g., polyglutamated[6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF (e.g.,polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF(e.g., polyglutamated [6S]-5-methyl-THF); (e) polyglutamatedTetrahydrofolate THF (e.g., polyglutamated [6S]-Tetrahydrofolate THF);(f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated[6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g.,polyglutamated [6S]-5-formimino-THF). In some embodiments, theadministered delivery vehicle comprises polyglutamated5,10-methylene-THF. In further embodiments, the administered deliveryvehicle comprises polyglutamated [6R]-5,10-methylene-THF. In otherembodiments, the administered delivery vehicle comprises polyglutamated[6R,S]-5,10-methylene-THF. In some embodiments, the administereddelivery vehicle comprises polyglutamated 5-methyl-THF. In furtherembodiments, the administered delivery vehicle comprises[6S]-5-methyl-THF. In other embodiments, the administered deliveryvehicle comprises [6R,S]-5-methyl-THF. In some embodiments, theadministered delivery vehicle comprises polyglutamated 5-formyl-THF. Infurther embodiments, the administered delivery vehicle comprisespolyglutamated [6S]-5-formyl-THF. In other embodiments, the administereddelivery vehicle comprises polyglutamated [6R,S]-5-formyl-THF.

In some embodiments, the cancer treated by one or more of the methodsdisclosed herein is a solid tumor lymphoma. Examples of solid tumorlymphoma include Hodgkin's lymphoma, Non-Hodgkin's lymphoma, and B celllymphoma.

In some embodiments, the cancer treated by one or more of the methodsdisclosed herein is bone cancer, brain cancer, breast cancer, colorectalcancer, connective tissue cancer, cancer of the digestive system,endometrial cancer, esophageal cancer, eye cancer, cancer of the headand neck, gastric cancer, intra-epithelial neoplasm, melanomaneuroblastoma, Non-Hodgkin's lymphoma, non-small cell lung cancer,prostate cancer, retinoblastoma, or rhabdomyosarcoma. In someembodiments, the cancer treated by one or more of the methods disclosedherein is selected from the group consisting of: lung cancer, pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, head and neckcancer, gastric cancer, gastrointestinal cancer, colorectal cancer,esophageal cancer, cervical cancer, liver cancer, kidney cancer, biliaryduct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, melanoma,myeloma, a leukemia and a lymphoma. In some embodiments, the cancertreated by one or more of the methods disclosed herein is selected fromthe group consisting of: colorectal cancer, breast cancer, gastriccancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lungcancer (e.g., non-small cell lung cancer and/or adenocarcinoma), headand neck cancer, ovarian cancer, gallbladder cancer, and basal cellcancer. In particular embodiments, the cancer treated by one or more ofthe methods disclosed herein is colorectal cancer

In some embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a compositioncomprising a delivery vehicle and polyglutamated alpha tetrahydrofolateto a subject having or at risk of having cancer. In some embodiments,the administered composition comprises a pegylated delivery vehicle. Insome embodiments, the administered composition comprises a targetingmoiety that has a specific affinity for an epitope of an antigen on thesurface of a target cell of interest such as a cancer cell. In someembodiments, the delivery vehicle comprises an antibody or an antigenbinding antibody fragment. In some embodiments, the composition isadministered to treat a cancer selected from the group consisting of:lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostatecancer, head and neck cancer, gastric cancer, gastrointestinal cancer,colorectal cancer, esophageal cancer, cervical cancer, liver cancer,kidney cancer, biliary duct cancer, gallbladder cancer, bladder cancer,sarcoma (e.g., osteosarcoma), brain cancer, central nervous systemcancer, melanoma, myeloma, a leukemia and a lymphoma. In someembodiments, the composition is administered to treat a cancer selectedfrom the group consisting of: colorectal cancer, breast cancer, gastriccancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lungcancer (e.g., non-small cell lung cancer and/or adenocarcinoma), headand neck cancer, ovarian cancer, gallbladder cancer, and basal cellcancer. In particular embodiments, the composition is administered totreat colorectal cancer. In some embodiments, the administeredcomposition contains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups.In some embodiments, the administered composition comprisestetraglutamated alpha tetrahydrofolate. In some embodiments, theadministered composition comprises pentaglutamated alphatetrahydrofolate. In other embodiments, the administered compositioncomprises hexaglutamated alpha tetrahydrofolate. In some embodiments,the administered composition comprises a αPTHF selected from: (a)polyglutamated 5-formyl-THF (e.g., polyglutamated [6S]-5-formyl-THF);(b) polyglutamated 10-formyl-THF (e.g., polyglutamated[6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF (e.g.,polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF(e.g., polyglutamated [6S]-5-methy 1-THF); (e) polyglutamatedTetrahydrofolate THF (e.g., polyglutamated [6S]-Tetrahydrofolate THF);(f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated[6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g.,polyglutamated [6S]-5-formimino-THF). In some embodiments, theadministered composition comprises polyglutamated 5,10-methylene-THF. Infurther embodiments, the administered composition comprisespolyglutamated [6R]-5,10-methylene-THF. In other embodiments, theadministered delivery vehicle comprises polyglutamated[6R,S]-5,10-methylene-THF. In some embodiments, the administeredcomposition polyglutamated 5-methyl-THF. In further embodiments, theadministered composition comprises [6S]-5-methyl-THF. In otherembodiments, the administered composition comprises [6R,S]-5-methyl-THF.In some embodiments, the administered composition polyglutamated5-formyl-THF. In further embodiments, the administered compositioncomprises polyglutamated [6S]-5-formyl-THF. In other embodiments, theadministered composition comprises polyglutamated [6R,S]-5-formyl-THF.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomalcomposition comprising liposomes that contain polyglutamated alphatetrahydrofolate (e.g., Lp-αPTHF, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF,TLp-αPTHF or TPLp-αPTHF) to a subject having or at risk of havingcancer. In some embodiments, the liposomal composition is administeredto treat a cancer selected from the group consisting of: lung cancer,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, headand neck cancer, gastric cancer, gastrointestinal cancer, colorectalcancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer,biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, melanoma,myeloma, a leukemia and a lymphoma. In some embodiments, the liposomalcomposition is administered to treat a cancer selected from the groupconsisting of: colorectal cancer, breast cancer, gastric cancer (e.g.,stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g.,non-small cell lung cancer and/or adenocarcinoma), head and neck cancer,ovarian cancer, gallbladder cancer, and basal cell cancer. In particularembodiments, the liposomal composition is administered to treatcolorectal cancer. In some embodiments, the administered liposomalcomposition comprises pegylated liposomes (e.g., PLp-αPTHF, NTPLp-αPTHF,or TPLp-αPTHF). In some embodiments, a liposome of the administeredliposomal composition comprises a αPTHF containing 4, 5, 2-10, 4-6, ormore than 5, glutamyl groups. In some embodiments, a liposome of theadministered liposomal composition comprise tetraglutamated alphatetrahydrofolate. In some embodiments, a liposome of the administeredliposomal composition comprise pentaglutamated alpha tetrahydrofolate.In other embodiments, a liposome of the administered liposomalcomposition comprises hexaglutamated alpha tetrahydrofolate. In someembodiments, a liposome of the liposomal composition comprises a αPTHFselected from: (a) polyglutamated 5-formyl-THF (e.g., polyglutamated[6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF (e.g.,polyglutamated [6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF(e.g., polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF); (e)polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, a liposome of the liposomal composition comprisespolyglutamated 5,10-methylene-THF. In further embodiments, a liposome ofthe liposomal composition comprises polyglutamated[6R]-5,10-methylene-THF. In other embodiments, a liposome of theliposomal composition comprises polyglutamated[6R,S]-5,10-methylene-THF. In some embodiments, a liposome of theliposomal composition comprises polyglutamated 5-methyl-THF. In furtherembodiments, a liposome of the liposomal composition comprises[6S]-5-methyl-THF. In other embodiments, a liposome of the liposomalcomposition comprises [6R,S]-5-methyl-THF. In some embodiments, aliposome of the liposomal composition comprises polyglutamated5-formyl-THF. In further embodiments, a liposome of the liposomalcomposition comprises polyglutamated [6S]-5-formyl-THF. In otherembodiments, a liposome of the liposomal composition comprisespolyglutamated [6R,S]-5-formyl-THF.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomalcomposition that comprises targeted liposomes (e.g., TLp-αPTHF orTPLp-αPTHF) to a subject having or at risk of having cancer, wherein theliposomal composition comprises liposomes that comprise polyglutamatedalpha tetrahydrofolate (Lp-αPTHF) and further comprise a targetingmoiety having a specific affinity for a surface antigen (epitope) on thecancer. In some embodiments, the liposomal composition is administeredto treat a cancer selected from the group consisting of: lung cancer,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, headand neck cancer, gastric cancer, gastrointestinal cancer, colorectalcancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer,biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, melanoma,myeloma, a leukemia and a lymphoma. In some embodiments, the liposomalcomposition is administered to treat a cancer selected from the groupconsisting of: colorectal cancer, breast cancer, gastric cancer (e.g.,stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g.,non-small cell lung cancer and/or adenocarcinoma), head and neck cancer,ovarian cancer, gallbladder cancer, and basal cell cancer. In particularembodiments, the liposomal composition is administered to treatcolorectal cancer. In some embodiments, the administered liposomalcomposition comprises pegylated liposomes (e.g., TPLp-αPTHF). In someembodiments, a liposome of the liposomal composition comprises Lpolyglutamated alpha tetrahydrofolate. In some embodiments, liposomes ofthe administered liposomal composition comprise a αPTHF containing 4, 5,2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, aliposome of the administered liposomal composition comprisestetraglutamated alpha tetrahydrofolate. In some embodiments, a liposomeof the administered liposomal composition comprises pentaglutamatedalpha tetrahydrofolate. In other embodiments, a liposome of theadministered liposomal composition comprises hexaglutamated alphatetrahydrofolate. In some embodiments, an administered liposomecomprises a αPTHF selected from: (a) polyglutamated 5-formyl-THF (e.g.,polyglutamated [6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF(e.g., polyglutamated [6R]-10-formyl-THF); (c) polyglutamated5,10-methenyl-THF (e.g., polyglutamated [6R]-5,10-methenyl-THF); (d)polyglutamated 5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF);(e) polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, an administered liposome comprises polyglutamated5,10-methylene-THF. In further embodiments, an administered liposomecomprises polyglutamated [6R]-5,10-methylene-THF. In other embodiments,an administered liposome comprises polyglutamated[6R,S]-5,10-methylene-THF. In some embodiments, an administered liposomecomprises polyglutamated 5-methyl-THF. In further embodiments, anadministered liposome comprises [6S]-5-methyl-THF. In other embodiments,an administered liposome comprises [6R,S]-5-methyl-THF. In someembodiments, an administered liposome comprises polyglutamated5-formyl-THF. In further embodiments, an administered liposome comprisespolyglutamated [6S]-5-formyl-THF. In other embodiments, an administeredliposome comprises polyglutamated [6R,S]-5-formyl-THF.

In further embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomalcomposition that contains targeted liposomes (e.g., TLp-αPTHF orTPLp-αPTHF) to a subject having or at risk of having a cancer thatexpresses folate receptor on its cell surface, wherein the liposomalcomposition comprises liposomes that comprise (a) polyglutamated alphatetrahydrofolate (αPTHF) and (b) a targeting moiety that has specificbinding affinity for the folate receptor. In some embodiments, theadministered liposomal composition comprises pegylated liposomes (e.g.,TPLp-αPTHF). In some embodiments, the targeting moiety has a specificbinding affinity for folate receptor alpha (FR-α), folate receptor beta(FR-β), and/or folate receptor delta (FR-δ). In some embodiments, thetargeting moiety has a specific binding affinity for folate receptoralpha (FR-α), folate receptor beta (FR-β), and/or folate receptor delta(FR-δ). In some embodiments, the targeting moiety has a specific bindingaffinity for folate receptor alpha (FR-late receptor beta (FR-β), and/orfolate receptor delta (FR-δ). In some embodiments, the targeting moietyhas a specific binding affinity for folate receptor alpha (FR-α) andfolate receptor beta (FR-β). In some embodiments, the liposomalcomposition is administered to treat a cancer selected from the groupconsisting of: lung cancer, pancreatic, breast cancer, ovarian cancer,lung cancer, prostate cancer, head and neck cancer, gastric cancer,gastrointestinal cancer, colon cancer, esophageal cancer, cervicalcancer, kidney cancer, biliary duct cancer, gallbladder cancer, and ahematologic malignancy In some embodiments, a liposome of theadministered liposomal composition comprise a αPTHF containing 4, 5,2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, aliposome of the administered liposomal composition comprisestetraglutamated alpha tetrahydrofolate. In some embodiments, a liposomeof the administered liposomal composition comprises pentaglutamatedalpha tetrahydrofolate. In other embodiments, a liposome of theadministered liposomal composition comprises hexaglutamated alphatetrahydrofolate. In some embodiments, an administered liposomecomprises a αPTHF selected from: (a) polyglutamated 5-formyl-THF (e.g.,polyglutamated [6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF(e.g., polyglutamated [6R]-10-formyl-THF); (c) polyglutamated5,10-methenyl-THF (e.g., polyglutamated [6R]-5,10-methenyl-THF); (d)polyglutamated 5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF);(e) polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, an administered liposome comprises polyglutamated5,10-methylene-THF. In further embodiments, an administered liposomecomprises polyglutamated [6R]-5,10-methylene-THF. In other embodiments,an administered liposome comprises polyglutamated[6R,S]-5,10-methylene-THF. In some embodiments, an administered liposomecomprises polyglutamated 5-methyl-THF. In further embodiments, anadministered liposome comprises [6S]-5-methyl-THF. In other embodiments,an administered liposome comprises [6R,S]-5-methyl-THF. In someembodiments, an administered liposome comprises polyglutamated5-formyl-THF. In further embodiments, an administered liposome comprisespolyglutamated [6S]-5-formyl-THF. In other embodiments, an administeredliposome comprises polyglutamated [6R,S]-5-formyl-THF.

In some embodiments, the disclosure provides a method for treating adisorder of the immune system (e.g., an autoimmune disease such asinflammation and rheumatoid arthritis) that comprises administering aneffective amount of a delivery vehicle (e.g., antibody or liposome)comprising polyglutamated alpha tetrahydrofolate (e.g., a αPTHFdisclosed herein) to a subject having or at risk of having a disorder ofthe immune system. In some embodiments, the delivery vehicle is anantibody (e.g., a full-length IgG antibody, a bispecific antibody, or ascFv). In some embodiments, the delivery vehicle is a liposome (e.g., anLp-αPTHF such as, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, orTPLp-αPTHF). In some embodiments, the administered delivery vehicle ispegylated. In some embodiments, the administered delivery vehicle is notpegylated. In additional embodiments, the administered delivery vehiclecomprises a targeting moiety that has a specific affinity for an epitopeof an antigen on the surface of an immune cell associated with adisorder of the immune system. In some embodiments, the targeting moietyis an antibody or an antigen binding antibody fragment. In someembodiments, the administered delivery vehicle comprises αPTHFcontaining 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the administered delivery vehicle comprises tetraglutamatedalpha tetrahydrofolate. In some embodiments, the administered deliveryvehicle comprises pentaglutamated alpha tetrahydrofolate. In otherembodiments, the administered delivery vehicle comprises hexaglutamatedalpha tetrahydrofolate. In some embodiments, the administered deliveryvehicle comprises a αPTHF selected from: (a) polyglutamated 5-formyl-THF(e.g., polyglutamated [6S]-5-formyl-THF); (b) polyglutamated10-formyl-THF (e.g., polyglutamated [6R]-10-formyl-THF); (c)polyglutamated 5,10-methenyl-THF (e.g., polyglutamated[6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF (e.g.,polyglutamated [6S]-5-methyl-THF); (e) polyglutamated TetrahydrofolateTHF (e.g., polyglutamated [6S]-Tetrahydrofolate THF); (f) polyglutamated5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and(g) polyglutamated 5-formimino-THF (e.g., polyglutamated[6S]-5-formimino-THF). In some embodiments, the administered deliveryvehicle comprises polyglutamated 5,10-methylene-THF. In furtherembodiments, the administered delivery vehicle comprises polyglutamated[6R]-5,10-methylene-THF. In other embodiments, the administered deliveryvehicle comprises polyglutamated [6R,S]-5,10-methylene-THF. In someembodiments, the administered delivery vehicle comprises polyglutamated5-methyl-THF. In further embodiments, the administered delivery vehiclecomprises [6S]-5-methyl-THF. In other embodiments, the administereddelivery vehicle comprises [6R,S]-5-methyl-THF. In some embodiments, theadministered delivery vehicle comprises polyglutamated 5-formyl-THF. Infurther embodiments, the administered delivery vehicle comprisespolyglutamated [6S]-5-formyl-THF. In other embodiments, the administereddelivery vehicle comprises polyglutamated [6R,S]-5-formyl-THF. In someembodiments, the autoimmune disease is inflammation and rheumatoidarthritis.

In some embodiments, the disclosure provides a method for treating aninfectious disease (e.g., HIV) that comprises administering an effectiveamount of a delivery vehicle (e.g., antibody or liposome) comprisingpolyglutamated alpha tetrahydrofolate (e.g., a αPTHF disclosed herein)to a subject having or at risk of having an infectious disease. In someembodiments, the delivery vehicle is an antibody (e.g., a full-lengthIgG antibody, a bispecific antibody, or a scFv). In some embodiments,the delivery vehicle is a liposome (e.g., an Lp-αPTHF such as,PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF). In someembodiments, the administered delivery vehicle is pegylated. In someembodiments, the administered delivery vehicle is not pegylated. Inadditional embodiments, the administered delivery vehicle comprises atargeting moiety that has a specific affinity for an epitope of anantigen on the surface of a pathogen associated with an infectiousdisease. In some embodiments, the targeting moiety is an antibody or anantigen binding antibody fragment. In some embodiments, the administereddelivery vehicle comprises αPTHF containing 4, 5, 2-10, 4-6, or morethan 5, glutamyl groups. In some embodiments, the administered deliveryvehicle comprises tetraglutamated alpha tetrahydrofolate. In someembodiments, the administered delivery vehicle comprises pentaglutamatedalpha tetrahydrofolate. In other embodiments, the administered deliveryvehicle comprises hexaglutamated alpha tetrahydrofolate. In someembodiments, the administered delivery vehicle comprises a αPTHFselected from: (a) polyglutamated 5-formyl-THF (e.g., polyglutamated[6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF (e.g.,polyglutamated [6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF(e.g., polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF); (e)polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In someembodiments, the administered delivery vehicle comprises polyglutamated5,10-methylene-THF. In further embodiments, the administered deliveryvehicle comprises polyglutamated [6R]-5,10-methylene-THF. In otherembodiments, the administered delivery vehicle comprises polyglutamated[6R,S]-5,10-methylene-THF. In some embodiments, the administereddelivery vehicle comprises polyglutamated 5-methyl-THF. In furtherembodiments, the administered delivery vehicle comprises[6S]-5-methyl-THF. In other embodiments, the administered deliveryvehicle comprises [6R,S]-5-methyl-THF. In some embodiments, theadministered delivery vehicle comprises polyglutamated 5-formyl-THF. Infurther embodiments, the administered delivery vehicle comprisespolyglutamated [6S]-5-formyl-THF. In other embodiments, the administereddelivery vehicle comprises polyglutamated [6R,S]-5-formyl-THF.

In some embodiments, the administered delivery vehicle is a liposome. Infurther embodiments, the liposome is pegylated. In additionalembodiments, the delivery vehicle comprises a targeting moiety on itssurface that specifically binds an antigen on the surface of a targetcell of interest. In further embodiments, the delivery vehicle comprisesa targeting moiety that has specific affinity for an epitope of a cellsurface antigen selected from the group consisting of: GONMB, TACSTD2(TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-α,folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1,mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b,CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16,Tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin, fibronectinextra-domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin,endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2,FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7,FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15,CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38,CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138,cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA1,EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3,EphB4, EphB6, an integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA,c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB,TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the delivery vehicle comprises a targeting moietythat has specific affinity for an epitope on a cell surface antigen(s)derived from, or determined to be expressed on, a specific subject'scancer (tumor) such as a neoantigen.

In further embodiments, the delivery vehicle is a liposome, and theliposome comprises a targeting moiety that has specific affinity for anepitope of a cell surface antigen selected from the group consisting of:GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin,fibronectin extra-domain B (ED-B), VEGFR2 (CD309), tenascin, collagenIV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeon a cell surface antigen(s) derived from, or determined to be expressedon, a specific subject's cancer (tumor) such as a neoantigen.

In some embodiments, the disclosure provides for the use of acomposition comprising a polyglutamated alpha tetrahydrofolate formanufacture of a medicament for treatment of a hyperproliferativedisease. In some embodiments, the polyglutamated alpha tetrahydrofolatecomprise 5 or more glutamyl groups. In some embodiments, thepolyglutamated alpha tetrahydrofolate is pentaglutamated orhexaglutamated. In some embodiments, the polyglutamated alphatetrahydrofolate is polyglutamated tetrahydrofolate (THF),tetrahydrofolate (THF). In some embodiments, the polyglutamated alphatetrahydrofolate is in a liposome. In some embodiments, thehyperproliferative disease is cancer. In some embodiments, the cancer isselected from the group consisting of: lung (e.g., non-small lungcancer), pancreatic, breast cancer, ovarian, lung, prostate, head andneck, gastric, gastrointestinal, colon, esophageal, cervical, kidney,biliary duct, gallbladder, and a hematologic malignancy. In someembodiments, the cancer is pancreatic cancer. In some embodiments, thecancer is breast cancer. In some embodiments, the cancer is pancreaticcancer. In some embodiments, the cancer is triple negative breastcancer. In some embodiments, the cancer is lung cancer. In someembodiments, the cancer is non-small cell lung cancer. In someembodiments, the cancer is leukemia or lymphoma. In some embodiments,the hyperproliferative disease is an autoimmune disease. In someembodiments, the hyperproliferative disease is inflammation andrheumatoid arthritis.

The disclosed methods can practiced in any subject that is likely tobenefit from delivery of compositions contemplated herein (e.g.,polyglutamated alpha tetrahydrofolate compositions such as liposomecontaining a pentaglutamated alpha or hexaglutamated alpha THF).Mammalian subjects, and in particular, human subjects are preferred. Insome embodiments, the subjects also include animals such as householdpets (e.g., dogs, cats, rabbits, and ferrets), livestock or farm animals(e.g., cows, pigs, sheep, chickens and other poultry), horses such asthoroughbred horses, laboratory animals (e.g., mice, rats, and rabbits),and other mammals. In other embodiments, the subjects include fish andother aquatic species.

The subjects to whom the agents are delivered may be normal subjects.Alternatively the subject may have or be at risk of developing acondition that can be diagnosed or that can benefit from delivery of oneor more of the provided compositions. In some embodiments, suchconditions include cancer (e.g., solid tumor cancers or non-solid cancersuch as leukemias). In some embodiments, these conditions (e.g.,cancers) involve cells that express an antigen that can be specificallybound by a targeted pegylated liposomal polyglutamated alphatetrahydrofolate disclosed herein. In further embodiments, theseantigens specifically bind and internalize the targeted pegylatedliposomal polyglutamated alpha tetrahydrofolate into the cell. In someembodiments, the targeted pegylated liposomal polyglutamated alphatetrahydrofolate specifically binds a folate receptor (e.g., folatereceptor alpha (FR-α), folate receptor beta (FR-β) and folate receptordelta (FR-δ)) expressed on the surface of the cancer cell.

Tests for diagnosing the conditions that can be treated with theprovided compositions are known in the art and will be familiar to themedical practitioner. The determination of whether a cell type expressesfolate receptors can be made using commercially available antibodies.These laboratory tests include without limitation microscopic analyses,cultivation dependent tests (such as cultures), and nucleic aciddetection tests. These include wet mounts, stain-enhanced microscopy,immune microscopy (e.g., FISH), hybridization microscopy, particleagglutination, enzyme-linked immunosorbent assays, urine screeningtests, DNA probe hybridization, and serologic tests. The medicalpractitioner will generally also take a full history and conduct acomplete physical examination in addition to running the laboratorytests listed above.

A subject having a cancer can, for example, be a subject that hasdetectable cancer cells. A subject at risk of developing a cancer can,for example, be a subject that has a higher than normal probability ofdeveloping cancer. These subjects include, for instance, subjects havinga genetic abnormality that has been demonstrated to be associated with ahigher likelihood of developing a cancer, subjects having a familialdisposition to cancer, subjects exposed to cancer causing agents (e.g.,carcinogens) such as tobacco, asbestos, or other chemical toxins, andsubjects previously treated for cancer and in apparent remission.

In some embodiments, the disclosure provides methods for selectivelydeliver a folate receptor targeted pegylated liposomal polyglutamatedalpha tetrahydrofolate to a tumor cell expressing a folate receptor onits surface at a rate that is higher (e.g., at least two-fold greater,at least three-fold greater, at least four-fold greater, or at leastfive-fold greater, than a cell not expressing folate receptor on itscell surface). In some embodiments, the delivered pegylated liposomecomprises polyglutamated alpha THF. In some embodiments, the deliveredpegylated liposome comprises L-polyglutamated alpha THF. In someembodiments, the delivered pegylated liposome comprises D-polyglutamatedalpha THF.

i. Combination Therapy

In certain embodiments, the disclosure provides administering apolyglutamated alpha THF composition described herein in combinationwith at least one additional therapeutic agent. An additionaltherapeutic agent can be administered prior to, concurrently with,and/or subsequently to, administration of the polyglutamated alpha THFcomposition. The additional therapeutic agent can be associated with apolyglutamated alpha THF delivery vehicle (e.g., coencapsulated withpolyglutamated alpha THF in a liposome), present in a solutioncontaining a polyglutamated alpha THF delivery vehicle, or in a separateformulation from the composition containing the polyglutamated alpha THFcomposition. Pharmaceutical compositions comprising a polypeptide oragent and the additional therapeutic agent(s) are also provided. In someembodiments, the at least one additional therapeutic agent comprises 1,2, 3, or more additional therapeutic agents.

Combination therapy with two or more therapeutic agents often usesagents that work by different mechanisms of action, although this is notrequired. Combination therapy using agents with different mechanisms ofaction may result in additive or synergetic effects. Combination therapymay allow for a lower dose of each agent than is used in monotherapy,thereby reducing toxic side effects and/or increasing the therapeuticindex of the polypeptide or agent(s). Combination therapy may decreasethe likelihood that resistant cancer cells will develop. In someembodiments, combination therapy comprises a therapeutic agent thataffects the immune response (e.g., enhances or activates the response)and a therapeutic agent that affects (e.g., inhibits or kills) thetumor/cancer cells.

In some embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of apolyglutamated alpha tetrahydrofolate composition disclosed herein and abiologic. In some embodiments, the polyglutamated alpha tetrahydrofolateis administered in combination with a therapeutic antibody. In furtherembodiments, the polyglutamated alpha tetrahydrofolate is administeredin combination with an anti-CD antibody (e.g., rituximab) or an antibodythat binds an immune checkpoint protein (e.g., CTLA4, PD1, PDL1, andTIM3). In further embodiments, the polyglutamated alpha tetrahydrofolateis administered in combination with an fc-fusion protein (e.g.,entanercept).

In some embodiments, the disclosure provides a method for treatingdisorder of the immune system that comprises administering an effectiveamount of a polyglutamated alpha tetrahydrofolate composition disclosedherein and a biologic. In some embodiments, the polyglutamated alphatetrahydrofolate is administered in combination with a therapeuticantibody. In further embodiments, the polyglutamated alphatetrahydrofolate is administered in combination with an anti-TNFantibody (e.g., adalimumab). In some embodiments, the polyglutamatedalpha tetrahydrofolate is administered in combination with an fc-fusionprotein (e.g., entanercept).

In some embodiments, of the methods described herein, the combination ofa αPTHF compositions described herein and at least one additionaltherapeutic agent results in additive or synergistic results. In someembodiments, the combination therapy results in an increase in thetherapeutic index of the αPTHF or agent. In some embodiments, thecombination therapy results in an increase in the therapeutic index ofthe additional therapeutic agent(s). In some embodiments, thecombination therapy results in a decrease in the toxicity and/or sideeffects of the αPTHF or agent. In some embodiments, the combinationtherapy results in a decrease in the toxicity and/or side effects of theadditional therapeutic agent(s).

Therapeutic agents that may be administered in combination with theαPTHF compositions described herein include chemotherapeutic agents.Thus, in some embodiments, the methods or treatments described hereinfurther comprise administering at least one αPTHF composition describedherein in combination with a chemotherapeutic agent or in combinationwith a cocktail of chemotherapeutic agents. In some embodiments, theadministered αPTHF is selected from: (a) polyglutamated 5-formyl-THF(e.g., polyglutamated [6S]-5-formyl-THF); (b) polyglutamated10-formyl-THF (e.g., polyglutamated [6R]-10-formyl-THF); (c)polyglutamated 5,10-methenyl-THF (e.g., polyglutamated[6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF (e.g.,polyglutamated [6S]-5-methyl-THF); (e) polyglutamated tetrahydrofolate(e.g., polyglutamated [6S]-Tetrahydrofolate THF); (f) polyglutamated5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and(g) polyglutamated 5-formimino-THF (e.g., polyglutamated[6S]-5-formimino-THF). Treatment with a αPTHF composition can occurprior to, concurrently with, or subsequent to administration ofchemotherapies. Combined administration can include co-administration,either in a single pharmaceutical formulation or using separateformulations, or consecutive administration in either order butgenerally within a time period such that all active agents can exerttheir biological activities simultaneously. Preparation and dosingschedules for such chemotherapeutic agents can be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in The Chemotherapy Source Book, 4.sup.th Edition, 2008,M. C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, Pa.

Chemotherapeutic agents useful in the present invention include, but arenot limited to, alkylating agents such as thiotepa and cyclosphosphamide(cytotaxan); alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamime; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as tetrahydrofolate and5-fluorouracil (5-FU); folic acid analogs such as denopterin,tetrahydrofolate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine,floxuridine, 5-FU; androgens such as calusterone, dromostanolonepropionate, epitiostanol, mepitiostane, testolactone; anti-adrenals suchas aminoglutethimide, mitotane, trilostane; folic acid replenishers suchas folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (Ara-C); taxoids, such as paclitaxel (TAXOL®®) and docetaxel(TAXOTERE®); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;tetrahydrofolate; ibandronate; CPT11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine (XELODA); anti-hormonal agents such as, tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON);anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; and pharmaceutically acceptable salts, acids orderivatives of any of the above. In certain embodiments, the additionaltherapeutic agent is cisplatin. In certain embodiments, the additionaltherapeutic agent is carboplatin. In other embodiments, the additionaltherapeutic agent is oxaloplatin. In particular embodiments, theadditional therapeutic agent is a pyrimidine analog (e.g., afluorpyrimidine such as 5-fluorouracil (5-FU)).

In some embodiments, the disclosure also provides methods or treatmentsthat comprise administering αPTHF described herein and at least oneadditional therapeutic agent selected from: an anti-tubulin agent, anauristatin, a DNA minor groove binder, a DNA replication inhibitor, analkylating agent (e.g., platinum complexes such as cisplatin,mono(platinum), bis(platinum) and tri-nuclear platinum complexes andcarboplatin), an anthracycline, an antibiotic, an anti-folate (e.g., apolyglutamatable antifolate or a non polyglutamatable anti-folate), anantimitotic (e.g., a vinca alkaloid, such as vincristine, vinblastine,vinorelbine, or vindesine), radiation sensitizer, a steroid, a taxane, atopoisomerase inhibitor (e.g., doxorubicin HCl, daunorubicin citrate,mitoxantrone HCl, actinomycin D, etoposide, topotecan HCl, teniposide(VM-26), and irinotecan), an anti-metabolite, a chemotherapy sensitizer,a duocarmycin, an etoposide, a fluorinated pyrimidine, an ionophore, alexitropsin, a nitrosourea, a platinol, a purine antimetabolite, a PARPinhibitor, and a puromycin. In certain embodiments, the secondtherapeutic agent is an alkylating agent, an antimetabolite, anantimitotic, a topoisomerase inhibitor, or an angiogenesis inhibitor. Insome embodiments, the administered αPTHF is selected from: (a)polyglutamated 5-formyl-THF (e.g., polyglutamated [6S]-5-formyl-THF);(b) polyglutamated 10-formyl-THF (e.g., polyglutamated[6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF (e.g.,polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF(e.g., polyglutamated [6S]-5-methyl-THF); (e) polyglutamatedtetrahydrofolate (e.g., polyglutamated [6S]-Tetrahydrofolate THF); (f)polyglutamated 5,10-methylene-THF (e.g., polyglutamated[6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g.,polyglutamated [6S]-5-formimino-THF).

In particular embodiments, treatment methods provided herein compriseadministering a αPTHF composition described herein in combination withan antifolate. In some embodiments, the αPTHF composition contains 4, 5,2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, theαPTHF composition is selected from: (a) polyglutamated 5-formyl-THF(e.g., polyglutamated [6S]-5-formyl-THF); (b) polyglutamated10-formyl-THF (e.g., polyglutamated [6R]-10-formyl-THF); (c)polyglutamated 5,10-methenyl-THF (e.g., polyglutamated[6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF (e.g.,polyglutamated [6S]-5-methyl-THF); (e) polyglutamated TetrahydrofolateTHF (e.g., polyglutamated [6S]-Tetrahydrofolate THF); (f) polyglutamated5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and(g) polyglutamated 5-formimino-THF (e.g., polyglutamated[6S]-5-formimino-THF. In some embodiments, the administered αPTHFcomposition is encapsulated in a liposome. In some embodiments, theαPTHF composition is administered separately from the antifolate. Insome embodiments, the αPTHF composition is administered at the same time(e.g., concurrently or serially) as the antifolate. In some embodiments,the αPTHF and the antifolate are encapsulated in the same liposome.

In particular embodiments, treatment methods provided herein compriseadministering a αPTHF composition described herein in combination withmethotrexate or a methotrexate derivative or analog. In someembodiments, the αPTHF composition contains 4, 5, 2-10, 4-6, or morethan 5, glutamyl groups. In some embodiments, the αPTHF composition isselected from: (a) polyglutamated 5-formyl-THF (e.g., polyglutamated[6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF (e.g.,polyglutamated [6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF(e.g., polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated5-methyl-THF (e.g., polyglutamated [6S]-5-methyl-THF); (e)polyglutamated Tetrahydrofolate THF (e.g., polyglutamated[6S]-Tetrahydrofolate THF); (f) polyglutamated 5,10-methylene-THF (e.g.,polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF. In someembodiments, the administered αPTHF composition is encapsulated in aliposome. In some embodiments, the αPTHF composition is administeredseparately from the methotrexate. In some embodiments, the αPTHFcomposition is administered at the same time (e.g., concurrently orserially) as the methotrexate. In some embodiments, the αPTHF andmethotrexate are encapsulated in the same liposome.

In particular embodiments, the disclosure provides administeringpolyglutamated 5,10-methylene-THF in combination with methotrexate. Insome embodiments, polyglutamated [6R]-5,10-methylene-THF is administeredin combination with methotrexate or a methotrexate derivative or analog.In some embodiments, tetraglutamated [6R]-5,10-methylene-THF isadministered in combination with methotrexate. In some embodiments,pentaglutamated [6R]-5,10-methylene-THF is administered in combinationwith methotrexate. In some embodiments, hexaglutamated[6R]-5,10-methylene-THF is administered after administration ofmethotrexate (e.g., to reduce one or more methotrexate associatedtoxicities). In some embodiments, polyglutamated [6R]-5,10-methylene-THFis administered in combination with methotrexate. In some embodiments,polyglutamated [6R,S]-5,10-methylene-THF is administered in combinationwith methotrexate. In some embodiments, tetraglutamated[6R,S]-5,10-methylene-THF is administered in combination withmethotrexate. In some embodiments, pentaglutamated[6R,S]-5,10-methylene-THF is administered in combination withmethotrexate. In some embodiments, hexaglutamated[6R,S]-5,10-methylene-THF is administered after the administration ofmethotrexate (e.g., to reduce one or more methotrexate associatedtoxicities). In some embodiments, the administered polyglutamated5,10-methylene-THF is encapsulated in a liposome. In some embodiments,the polyglutamated 5,10-methylene-THF is administered separately fromthe methotrexate. In some embodiments, the polyglutamated5,10-methylene-THF is administered at the same time (e.g., concurrentlyor serially) as the methotrexate. In some embodiments, thepolyglutamated 5,10-methylene-THF and methotrexate are encapsulated inthe same liposome.

In particular embodiments, the disclosure provides administeringpolyglutamated 5-methyl-THF in combination with methotrexate or amethotrexate derivative or analog. In some embodiments, polyglutamated[6S]-5-methyl-THF is administered in combination with methotrexate. Insome embodiments, tetraglutamated [6S]-5-methyl-THF is administered incombination with methotrexate. In some embodiments, pentaglutamated[6S]-5-methyl-THF is administered in combination with methotrexate. Insome embodiments, hexaglutamated [6S]-5-methyl-THF is administered afteradministration of methotrexate (e.g., to reduce one or more methotrexateassociated toxicities). In some embodiments, polyglutamated[6R,S]-5-methyl-THF is administered in combination with methotrexate. Insome embodiments, tetraglutamated [6R,S]-5-methyl-THF is administered incombination with methotrexate. In some embodiments, pentaglutamated[6R,S]-5-methyl-THF is administered in combination with methotrexate. Insome embodiments, hexaglutamated [6R,S]-5-methyl-THF is administeredafter administration of methotrexate (e.g., to reduce one or moremethotrexate associated toxicities). In some embodiments, thepolyglutamated 5-methyl-THF is encapsulated in a liposome. In someembodiments, the polyglutamated 5-methyl-THF is administered separatelyfrom the methotrexate. In some embodiments, the polyglutamated5-methyl-THF is administered at the same time (e.g., concurrently orserially) as the methotrexate. In some embodiments, the polyglutamated5-methyl-THF composition and methotrexate are encapsulated in the sameliposome.

In particular embodiments, the disclosure provides administeringpolyglutamated 5-formyl-THF in combination with methotrexate. In someembodiments, polyglutamated [6S]-5-formyl-THF is administered incombination with methotrexate. In some embodiments, tetraglutamated[6S]-5-formyl-THF is administered in combination with methotrexate. Insome embodiments, pentaglutamated [6S]-5-formyl-THF is administered incombination with methotrexate. In some embodiments, hexaglutamated[6S]-5-formyl-THF is administered after administration of methotrexate(e.g., to reduce one or more methotrexate associated toxicities). Insome embodiments, polyglutamated [6R,S]-formyl-THF is administered incombination with methotrexate. In some embodiments, tetraglutamated[6R,S]-5-formyl-THF is administered in combination with methotrexate. Insome embodiments, pentaglutamated [6R,S]-5-formyl-THF is administered incombination with methotrexate. In some embodiments, hexaglutamated[6R,S]-5-formyl-THF is administered after administration of methotrexate(e.g., to reduce one or more methotrexate associated toxicities). Insome embodiments, the polyglutamated 5-methyl-THF is encapsulated in aliposome. In some embodiments, the polyglutamated 5-methyl-THF isadministered separately from the methotrexate. In some embodiments, thepolyglutamated 5-methyl-THF is administered at the same time (e.g.,concurrently or serially) as the methotrexate. In some embodiments, thepolyglutamated 5-methyl-THF composition and methotrexate areencapsulated in the same liposome.

In some embodiments, the disclosure provides administering αPTHF incombination with an antimetabolite. In some embodiments, the αPTHF isadministered separately from the antimetabolite. In some embodiments,the αPTHF is administered at the same time (e.g., concurrently orserially) as the antimetabolite. In some embodiments, the αPTHF andantimetabolite are encapsulated in the same liposome. In someembodiments, the αPTHF is administered in combination with a pyrimidineanalog. In some embodiments, the administered αPTHF contains 4, 5, 2-10,4-6, or more than 5, glutamyl groups. In some embodiments, theadministered αPTHF composition is selected from: (a) polyglutamated5-formyl-THF (e.g., polyglutamated [6S]-5-formyl-THF); (b)polyglutamated 10-formyl-THF (e.g., polyglutamated [6R]-10-formyl-THF);(c) polyglutamated 5,10-methenyl-THF (e.g., polyglutamated[6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF (e.g.,polyglutamated [6S]-5-methyl-THF); (e) polyglutamated TetrahydrofolateTHF (e.g., polyglutamated [6S]-Tetrahydrofolate THF); (f) polyglutamated5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and(g) polyglutamated 5-formimino-THF (e.g., polyglutamated[6S]-5-formimino-THF. In some embodiments, the αPTHF is administered incombination with a pyrimidine analog selected from the group consistingof ancitabine, azacitidine, 6-azauridine, carmofur, cytosinearabinoside, dideoxyuridine, doxifluridine, enocitabine, floxuridine,and 5-FU. In some embodiments, the αPTHF is administered separately fromthe pyrimidine analog. In some embodiments, the αPTHF is administered atthe same time (e.g., concurrently or serially) as the pyrimidine analog.In some embodiments, the αPTHF and a pyrimidine analog are encapsulatedin the same liposome. In particular embodiments, the αPTHF isadministered in combination with 5-FU. In some embodiments, the αPTHF isadministered separately from the 5-FU. In some embodiments, the αPTHF isadministered at the same time (e.g., concurrently or serially) as the5-FU. In some embodiments, the αPTHF and 5-FU are encapsulated in thesame liposome.

In some embodiments, the disclosure provides administeringpolyglutamated 5,10-methylene-THF in combination with an antimetabolite.In some embodiments, the administered polyglutamated 5,10-methylene-THF(e.g., [6R]-5,10-methylene-THF or [6R,S]-5,10-methylene-THF) contains 4,5, 2-10, 4-6, or more than 5, glutamyl groups. In some embodiments,tetraglutamated 5,10-methylene-THF (e.g., [6R]-5,10-methylene-THF) isadministered in combination with the antimetabolite. In someembodiments, pentaglutamated 5,10-methylene-THF (e.g.,[6R]-5,10-methylene-THF) is administered in combination with theantimetabolite. In some embodiments, hexaglutamated 5,10-methylene-THF(e.g., [6R]-5,10-methylene-THF) is administered in combination with theantimetabolite. In some embodiments, polyglutamated[6R]-5,10-methylene-THF is administered in combination with theantimetabolite. In some embodiments, polyglutamated[6R,S]-5,10-methylene-THF is administered in combination with theantimetabolite. In some embodiments, the polyglutamated5,10-methylene-THF is administered separately from the antimetabolite.In some embodiments, the polyglutamated 5,10-methylene-THF isadministered at the same time (e.g., concurrently or serially) as theantimetabolite. In some embodiments, the polyglutamated5,10-methylene-THF and antimetabolite are encapsulated in the sameliposome. In some embodiments the polyglutamated 5,10-methylene-THF isadministered in combination with a pyrimidine analog. In someembodiments, the polyglutamated 5,10-methylene-THF is administered incombination with a pyrimidine analog selected from the group consistingof ancitabine, azacitidine, 6-azauridine, carmofur, cytosinearabinoside, dideoxyuridine, doxifluridine, enocitabine, floxuridine,and 5-FU. In some embodiments, polyglutamated [6R]-5,10-methylene-THF isadministered in combination with the pyrimidine analog. In someembodiments, polyglutamated [6R,S]-5,10-methylene-THF is administered incombination with the pyrimidine analog. In particular embodiments, thepolyglutamated 5,10-methylene-THF is administered in combination with5-FU. In some embodiments, polyglutamated [6R]-5,10-methylene-THF isadministered in combination with 5-FU. In some embodiments,polyglutamated [6R,S]-5,10-methylene-THF is administered in combinationwith 5-FU. In some embodiments, the polyglutamated 5,10-methylene-THF isadministered separately from the pyrimidine analog (e.g., 5-FU,capecitabine, or tegafur). In some embodiments, the polyglutamated5,10-methylene-THF is administered at the same time (e.g., concurrentlyor serially) as the pyrimidine analog (e.g., 5-FU, capecitabine, ortegafur). In some embodiments, the polyglutamated 5,10-methylene-THF anda pyrimidine analog (e.g., 5-FU, capecitabine, or tegafur) areencapsulated in the same liposome. In particular embodiments, thepolyglutamated 5,10-methylene-THF is administered in combination with5-FU. In some embodiments, the polyglutamated 5,10-methylene-THF isadministered separately from the 5-FU. In some embodiments, thepolyglutamated 5,10-methylene-THF is administered at the same time(e.g., concurrently or serially) as the 5-FU. In some embodiments, thepolyglutamated 5,10-methylene-THF and 5-FU are encapsulated in the sameliposome.

In some embodiments, the disclosure provides administeringpolyglutamated 5-methyl-THF in combination with an antimetabolite. Insome embodiments, the administered polyglutamated 5-methyl-THF (e.g.,[6S]-5-methyl-THF or [6R,S]-5-methyl-THF) contains 4, 5, 2-10, 4-6, ormore than 5, glutamyl groups. In some embodiments, tetraglutamated5-methyl-THF (e.g., [6S]-5-methyl-THF) is administered in combinationwith the antimetabolite. In some embodiments, pentaglutamated5-methyl-THF (e.g., [6S]-5-methyl-THF) is administered in combinationwith the antimetabolite. In some embodiments, hexaglutamated5-methyl-THF (e.g., [6S]-5-methyl-THF) is administered in combinationwith the antimetabolite. In some embodiments, polyglutamated[6S]-5-methyl-THF is administered in combination with theantimetabolite. In some embodiments, polyglutamated [6R,S]-5-methyl-THFis administered in combination with the antimetabolite. In someembodiments, the polyglutamated 5-methyl-THF is administered separatelyfrom the antimetabolite. In some embodiments, the polyglutamated5-methyl-THF is administered at the same time (e.g., concurrently orserially) as the antimetabolite. In some embodiments, the polyglutamated5-methyl-THF and antimetabolite are encapsulated in the same liposome.In some embodiments the polyglutamated 5-methyl-THF is administered incombination with a pyrimidine analog. In some embodiments, thepolyglutamated 5-methyl-THF is administered in combination with apyrimidine analog selected from the group consisting of ancitabine,azacitidine, 6-azauridine, carmofur, cytosine arabinoside,dideoxyuridine, doxifluridine, enocitabine, floxuridine, and 5-FU. Insome embodiments, polyglutamated [6S]-5-methyl-THF is administered incombination with the pyrimidine analog. In some embodiments,polyglutamated [6R,S]-5-methyl-THF is administered in combination withthe pyrimidine analog. In particular embodiments, the polyglutamated5-methyl-THF is administered in combination with 5-FU. In someembodiments, polyglutamated [6S]-5-methyl-THF is administered incombination with 5-FU. In some embodiments, polyglutamated[6R,S]-5-methyl-THF is administered in combination with 5-FU. In someembodiments, the polyglutamated 5-methyl-THF is administered separatelyfrom the pyrimidine analog (e.g., 5-FU, capecitabine, or tegafur). Insome embodiments, the polyglutamated 5-methyl-THF is administered at thesame time (e.g., concurrently or serially) as the pyrimidine analog(e.g., 5-FU, capecitabine, or tegafur),In some embodiments, thepolyglutamated 5-methyl-THF and a pyrimidine analog (e.g., 5-FU,capecitabine, or tegafur) are encapsulated in the same liposome. In someembodiments, the polyglutamated 5-methyl-THF is administered separatelyfrom the 5-FU. In some embodiments, the polyglutamated 5-methyl-THF isadministered at the same time (e.g., concurrently or serially) as the5-FU. In some embodiments, the polyglutamated 5-methyl-THF and 5-FU areencapsulated in the same liposome.

In some embodiments, the disclosure provides administeringpolyglutamated 5-formyl-THF in combination with an antimetabolite. Insome embodiments, the administered polyglutamated 5-formyl-THF (e.g.,[6S]-5-formyl-THF or [6R,S]-5-formyl-THF) contains 4, 5, 2-10, 4-6, ormore than 5, glutamyl groups. In some embodiments, tetraglutamated5-formyl-THF (e.g., [6S]-5-formyl-THF) is administered in combinationwith the antimetabolite. In some embodiments, pentaglutamated5-formyl-THF (e.g., [6S]-5-formyl-THF) is administered in combinationwith the antimetabolite. In some embodiments, hexaglutamated5-formyl-THF (e.g., [6S]-5-formyl-THF) is administered in combinationwith the antimetabolite. In some embodiments, polyglutamated[6S]-5-formyl-THF is administered in combination with theantimetabolite. In some embodiments, polyglutamated [6R,S]-5-formyl-THFis administered in combination with the antimetabolite. In someembodiments, the polyglutamated 5-formyl-THF is administered separatelyfrom the antimetabolite. In some embodiments, the polyglutamated5-formyl-THF is administered at the same time (e.g., concurrently orserially) as the antimetabolite. In some embodiments, the polyglutamated5-formyl-THF and antimetabolite are encapsulated in the same liposome.

In some embodiments the polyglutamated 5-formyl-THF THF (e.g.,[6S]-5-formyl-THF or [6R,S]-5-formyl-THF) is administered in combinationwith a pyrimidine analog. In some embodiments, the administeredpolyglutamated 5-formyl-THF contains 4, 5, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, tetraglutamated 5-formyl-THF(e.g., [6S]-5-formyl-THF) is administered in combination with thepyrimidine analog. In some embodiments, pentaglutamated 5-formyl-THF(e.g., [6S]-5-formyl-THF) is administered in combination with thepyrimidine analog. In some embodiments, hexaglutamated 5-formyl-THF(e.g., [6S]-5-formyl-THF) is administered in combination with thepyrimidine analog. In some embodiments, the polyglutamated 5-formyl-THFis administered in combination with a pyrimidine analog selected fromthe group consisting of ancitabine, azacitidine, 6-azauridine, carmofur,cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine,floxuridine, and 5-FU. In some embodiments, the administered pyrimidineanalog is a fluoropyrimidine. In some embodiments, the fluoropyrimidineis selected from capecitabine, tegafur (e.g., tegafur with or withouturacil, gimeracil and/or oteracil), and 5-fluorouracil (5-FU). In someembodiments, the administered fluoropyrimidine is 5-FU. In someembodiments, the administered fluoropyrimidine is capecitabine. In someembodiments, the administered fluoropyrimidine is tegafur with orwithout uracil, gimeracil and/or oteracil. In some embodiments,polyglutamated [6S]-5-formyl-THF is administered in combination with thepyrimidine analog. In some embodiments, polyglutamated[6R,S]-5-formyl-THF is administered in combination with the pyrimidineanalog. In particular embodiments, the polyglutamated 5-formyl-THF isadministered in combination with 5-FU. In some embodiments,polyglutamated [6S]-5-formyl-THF is administered in combination with5-FU. In some embodiments, polyglutamated [6R,S]-5-formyl-THF isadministered in combination with 5-FU. In some embodiments, thepolyglutamated 5-formyl-THF is administered separately from thepyrimidine analog (e.g., 5-FU, capecitabine, or tegafur). In someembodiments, the polyglutamated 5-formyl-THF is administered at the sametime (e.g., concurrently or serially) as the pyrimidine analog (e.g.,5-FU, capecitabine, or tegafur). In some embodiments, the polyglutamated5-formyl-THF and a pyrimidine analog (e.g., 5-FU, capecitabine, ortegafur) are encapsulated in the same liposome. In particularembodiments, the polyglutamated 5-formyl-THF is administered incombination with 5-FU. In some embodiments, the polyglutamated5-formyl-THF is administered separately from the 5-FU. In someembodiments, the polyglutamated 5-formyl-THF is administered at the sametime (e.g., concurrently or serially) as the 5-FU. In some embodiments,the polyglutamated 5-formyl-THF and 5-FU are encapsulated in the sameliposome.

In some embodiments, the αPTHF is administered in combination with aplatinum-based agent (e.g., cisplatin, carboplatin, and oxaliplatin). Insome embodiments, the administered αPTHF contains 4, 5, 2-10, 4-6, ormore than 5, glutamyl groups. In some embodiments, tetraglutamated αPTHFis administered in combination with the platinum-based agent. In someembodiments, αPTHF is administered in combination with theplatinum-based agent. In some embodiments, hexaglutamated αPTHF isadministered in combination with the platinum-based agent. In someembodiments, the administered αPTHF is selected from: (a) polyglutamated5-formyl-THF (e.g., polyglutamated [6S]-5-formyl-THF); (b)polyglutamated 10-formyl-THF (e.g., polyglutamated [6R]-10-formyl-THF);(c) polyglutamated 5,10-methenyl-THF (e.g., polyglutamated[6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF (e.g.,polyglutamated [6S]-5-methyl-THF); (e) polyglutamated TetrahydrofolateTHF (e.g., polyglutamated [6S]-Tetrahydrofolate THF); polyglutamated5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and(g) polyglutamated 5-formimino-THF (e.g., polyglutamated[6S]-5-formimino-THF. In some embodiments, the αPTHF is administeredseparately from the platinum-based agent. In some embodiments, the αPTHFis administered at the same time (e.g., concurrently or serially) as theplatinum-based agent. In some embodiments, the αPTHF and platinum-basedagent are encapsulated in the same liposome. In particular embodiments,the αPTHF composition is administered in combination with oxaliplatin.In some embodiments, the αPTHF is administered at the same time (e.g.,concurrently or serially) as the oxaloplatin. In some embodiments, theαPTHF and oxaloplatin are encapsulated in the same liposome.

In some embodiments, αPTHF is administered in combination with aplatinum-based agent (e.g., cisplatin, carboplatin, and oxaliplatin) anda pyrimidine analog. In some embodiments, the administered αPTHFcontains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, administered αPTHF is tetraglutamated αPTHF. In someembodiments, administered αPTHF is pentaglutamated αPTHF. In someembodiments, administered αPTHF is hexaglutamated αPTHF. In someembodiments, the administered pyrimidine analog is selected from:ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside,di deoxyuridine, doxifluridine, enocitabine, floxuridine, and 5-FU. Insome embodiments, the administered pyrimidine analog is afluoropyrimidine. In some embodiments, the fluoropyrimidine is selectedfrom capecitabine, tegafur (e.g., tegafur with or without uracil,gimeracil and/or oteracil), and 5-fluorouracil (5-FU). In someembodiments, the administered fluoropyrimidine is 5-FU. In someembodiments, the administered fluoropyrimidine is capecitabine. In someembodiments, the administered fluoropyrimidine is tegafur with orwithout uracil, gimeracil and/or oteracil. In some embodiments, theadministered αPTHF is selected from: (a) polyglutamated 5-formyl-THF(e.g., polyglutamated [6S]-5-formyl-THF); (b) polyglutamated10-formyl-THF (e.g., polyglutamated [6R]-10-formyl-THF); (c)polyglutamated 5,10-methenyl-THF (e.g., polyglutamated[6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF (e.g.,polyglutamated [6S]-5-methyl-THF); (e) polyglutamated TetrahydrofolateTHF (e.g., polyglutamated [6S]-Tetrahydrofolate THF); (f) polyglutamated5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and(g) polyglutamated 5-formimino-THF (e.g., polyglutamated[6S]-5-formimino-THF. In some embodiments, the αPTHF is administeredseparately from the platinum-based agent and/or the pyrimidine analog.In some embodiments, the αPTHF is administered at the same time (e.g.,concurrently or serially) as the platinum-based agent and/or thepyrimidine analog. In some embodiments, the αPTHF is encapsulated in aliposome. In some embodiments, (a) the αPTHF and platinum-based agentare encapsulated in the same liposome, (b) the αPTHF and a pyrimidineanalog are encapsulated in the same liposome, (c) the platinum-basedagent, and a pyrimidine analog are encapsulated in the same liposome, or(d) the αPTHF, platinum-based agent, and a pyrimidine analog areencapsulated in the same liposome.

In particular embodiments, the αPTHF is administered in combination withoxaloplatin and 5-FU (e.g., FOLFOX therapy). In some embodiments, theadministered αPTHF contains 4, 5, 2-10, 4-6, or more than 5, glutamylgroups. In some embodiments, administered αPTHF is tetraglutamatedαPTHF. In some embodiments, administered αPTHF is pentaglutamated αPTHF.In some embodiments, administered αPTHF is hexaglutamated αPTHF. In someembodiments, the αPTHF is encapsulated in a liposome. In someembodiments, (a) the αPTHF and oxaloplatin are encapsulated in the sameliposome, (b) the αPTHF and 5-FU are encapsulated in the same liposome,(c) the oxaloplatin and 5-FU are encapsulated in the same liposome, or(d) the αPTHF, oxaloplatin, and 5-FU are encapsulated in the sameliposome. In additional embodiments, the αPTHF, oxaloplatin and 5-FU, isadministered to a subject having or at risk of having cancer. In someembodiments, the cancer is selected from: colorectal cancer, breastcancer, gastric cancer, pancreatic cancer, liver cancer, lung cancer(e.g., non-small cell lung cancer and adenocarcinoma), head and neckcancer, ovarian cancer, and basal cell cancer. In particularembodiments, the cancer is colorectal cancer. In further embodiments,αPTHF is administered in combination with oxaloplatin, 5-FU, and anantibody that binds VEGF (e.g., bevacizumab).

In some embodiments, polyglutamated 5,10-methylene-THF is administeredin combination with a platinum-based agent (e.g., cisplatin,carboplatin, and oxaliplatin). In some embodiments, the administered5,10-methylene-THF (e.g., [6R]-5,10-methylene-THF, or[6R,S]-5,10-methylene-THF) contains 4, 5, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, administered 5,10-methylene-THF(e.g., [6R]-5,10-methylene-THF) is tetraglutamated 5,10-methylene-THF.In some embodiments, administered 5,10-methylene-THF (e.g.,[6R]-5,10-methylene-THF) is pentaglutamated 5,10-methylene-THF. In someembodiments, administered 5,10-methylene-THF (e.g.,[6R]-5,10-methylene-THF) is hexaglutamated 5,10-methylene-THF. In someembodiments, polyglutamated [6R]-5,10-methylene-THF is administered incombination with a platinum-based agent. In some embodiments,polyglutamated [6R,S]-5,10-methylene-THF is administered in combinationwith a platinum-based agent. In some embodiments, the 5,10-methylene-THFis administered separately from the platinum-based agent. In someembodiments, the 5,10-methylene-THF is administered at the same time(e.g., concurrently or serially) as the platinum-based agent. In someembodiments, the 5,10-methylene-THF and platinum-based agent areencapsulated in the same liposome. In particular embodiments, the5,10-methylene-THF composition is administered in combination withoxaliplatin. In some embodiments, the 5,10-methylene-THF is administeredseparately from the oxaloplatin. In some embodiments, the5,10-methylene-THF is administered at the same time (e.g., concurrentlyor serially) as the oxaloplatin. In some embodiments, the5,10-methylene-THF and oxaloplatin are encapsulated in the sameliposome.

In some embodiments, 5,10-methylene-THF is administered in combinationwith a platinum-based agent (e.g., cisplatin, carboplatin, andoxaliplatin) and a pyrimidine analog. In some embodiments, theadministered 5,10-methylene-THF (e.g., [6R]-5,10-methylene-THF, or[6R,S]-5,10-methylene-THF) contains 4, 5, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, administered 5,10-methylene-THF(e.g., [6R]-5,10-methylene-THF) is tetraglutamated 5,10-methylene-THF.In some embodiments, administered 5,10-methylene-THF (e.g.,[6R]-5,10-methylene-THF) is pentaglutamated 5,10-methylene-THF. In someembodiments, administered 5,10-methylene-THF (e.g.,[6R]-5,10-methylene-THF) is hexaglutamated 5,10-methylene-THF. In someembodiments, the administered pyrimidine analog is selected from:ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside,dideoxyuridine, doxifluridine, enocitabine, floxuridine, and 5-FU. Insome embodiments, polyglutamated [6R]-5,10-methylene-THF is administeredin combination with a platinum-based agent (e.g., cisplatin,carboplatin, and oxaliplatin) and a pyrimidine analog. In someembodiments, polyglutamated [6R,S]-5,10-methylene-THF is administered incombination with a platinum-based agent (e.g., cisplatin, carboplatin,and oxaliplatin) and a pyrimidine analog. In some embodiments, the5,10-methylene-THF is administered separately from the platinum-basedagent and/or the pyrimidine analog. In some embodiments, the5,10-methylene-THF is administered at the same time (e.g., concurrentlyor serially) as the platinum-based agent and/or the pyrimidine analog.In some embodiments, the 5,10-methylene-THF is encapsulated in aliposome. In some embodiments, (a) the 5,10-methylene-THF andplatinum-based agent are encapsulated in the same liposome, (b) the5,10-methylene-THF and a pyrimidine analog are encapsulated in the sameliposome, (c) the platinum-based agent, and a pyrimidine analog areencapsulated in the same liposome, or (d) the 5,10-methylene-THF,platinum-based agent, and a pyrimidine analog are encapsulated in thesame liposome.

In particular embodiments, 5,10-methylene-THF is administered incombination with oxaloplatin and 5-FU. In some embodiments, administered5,10-methylene-THF (e.g., [6R]-5,10-methylene-THF, or[6RS]-5,10-methylene-THF) contains 4, 5, 2-10, 4-6, or more than 5,glutamyl groups. In some embodiments, administered 5,10-methylene-THF(e.g., [6R]-5,10-methylene-THF) is tetraglutamated 5,10-methylene-THF.In some embodiments administered 5,10-methylene-THF (e.g.,[6R]-5,10-methylene-THF) is pentaglutamated 5,10-methylene-THF. In someembodiments, the administered 5,10-methylene-THF (e.g.,[6R]-5,10-methylene-THF) is hexaglutamated 5,10-methylene-THF. In someembodiments, the 5,10-methylene-THF is encapsulated in a liposome. Insome embodiments, (a) the 5,10-methylene-THF and oxaloplatin areencapsulated in the same liposome, (b) the 5,10-methylene-THF and 5-FUare encapsulated in the same liposome, (c) the oxaloplatin and 5-FU areencapsulated in the same liposome, or (d) the 5,10-methylene-THF,oxaloplatin, and 5-FU are encapsulated in the same liposome. Inadditional embodiments, the 5,10-methylene-THF, oxaloplatin and 5-FU,are administered to a subject having or at risk of having cancer. Inadditional embodiments, the 5,10-methylene-THF, oxaloplatin and 5-FU,are administered to a subject having or at risk of having cancer. Insome embodiments, the cancer is selected from: colorectal cancer, breastcancer, gastric cancer (e.g., stomach cancer), pancreatic cancer, livercancer, lung cancer (e.g., non-small cell lung cancer and/oradenocarcinoma), head and neck cancer, ovarian cancer, gallbladdercancer, and basal cell cancer. In particular embodiments, the cancer iscolorectal cancer. In further embodiments, 5,10-methylene-THF isadministered in combination with oxaloplatin, 5-FU, and an antibody thatbinds VEGF (e.g., bevacizumab).

In some embodiments, polyglutamated 5-methyl-THF is administered incombination with a platinum-based agent (e.g., cisplatin, carboplatin,and oxaliplatin). In some embodiments, the administered 5-methyl-THF(e.g., [6S]-5-methyl-THF, or [6R,S]-5-methyl-THF) contains 4, 5, 2-10,4-6, or more than 5, glutamyl groups. In some embodiments, administered5-methyl-THF (e.g., [6S]-5-methyl-THF) is tetraglutamated 5-methyl-THF.In some embodiments administered 5-methyl-THF (e.g., [6S]-5-methyl-THF)is pentaglutamated 5-methyl-THF. In some embodiments, administered5-methyl-THF (e.g., [6S]-5-methyl-THF) is hexaglutamated 5-methyl-THF.In some embodiments, polyglutamated [6S]-5-methyl-THF is administered incombination with a platinum-based agent. In some embodiments,polyglutamated [6R,S]-5-methyl-THF is administered in combination with aplatinum-based agent. In some embodiments, the 5-methyl-THF isadministered separately from the platinum-based agent. In someembodiments, the 5-methyl-THF is administered at the same time as theplatinum-based agent. In some embodiments, the 5-methyl-THF andplatinum-based agent are encapsulated in the same liposome. Inparticular embodiments, the 5-methyl-THF composition is administered incombination with oxaliplatin. In some embodiments, the 5-methyl-THF isadministered separately from the oxaloplatin. In some embodiments, the5-methyl-THF is administered at the same time (e.g., concurrently orserially) as the oxaloplatin. In some embodiments, the 5-methyl-THF andoxaloplatin are encapsulated in the same liposome.

In some embodiments, 5-methyl-THF is administered in combination with aplatinum-based agent (e.g., cisplatin, carboplatin, and oxaliplatin) anda pyrimidine analog. In some embodiments, the administered 5-methyl-THF(e.g., [6S]-5-methyl-THF, or [6R,S]-5-methyl-THF) contains 4, 5, 2-10,4-6, or more than 5, glutamyl groups. In some embodiments, administered5-methyl-THF (e.g., [6S]-5-methyl-THF) is tetraglutamated 5-methyl-THF.In some embodiments administered 5-methyl-THF (e.g., [6S]-5-methyl-THF)is pentaglutamated 5-methyl-THF. In some embodiments, administered5-methyl-THF (e.g., [6S]-5-methyl-THF) is hexaglutamated 5-methyl-THF.In some embodiments, the administered pyrimidine analog is selectedfrom: ancitabine, azacitidine, 6-azauridine, carmofur, cytosinearabinoside, dideoxyuridine, doxifluridine, enocitabine, floxuridine,and 5-FU. In some embodiments, polyglutamated [6S]-5-methyl-THF isadministered in combination with a platinum-based agent (e.g.,cisplatin, carboplatin, and oxaliplatin) and a pyrimidine analog. Insome embodiments, polyglutamated [6R,S]-5-methyl-THF is administered incombination with a platinum-based agent (e.g., cisplatin, carboplatin,and oxaliplatin) and a pyrimidine analog. In some embodiments, the5-methyl-THF is administered separately from the platinum-based agentand/or the pyrimidine analog. In some embodiments, the 5-methyl-THF isadministered at the same time (e.g., concurrently or serially) as theplatinum-based agent and/or the pyrimidine analog. In some embodiments,the 5-methyl-THF is encapsulated in a liposome. In some embodiments, (a)the 5-methyl-THF and platinum-based agent are encapsulated in the sameliposome, (b) the 5-methyl-THF and a pyrimidine analog are encapsulatedin the same liposome, (c) the platinum-based agent, and a pyrimidineanalog are encapsulated in the same liposome, or (d) the 5-methyl-THF,platinum-based agent, and a pyrimidine analog are encapsulated in thesame liposome.

In particular embodiments, 5-methyl-THF is administered in combinationwith oxaloplatin and 5-FU. In some embodiments, the administered5-methyl-THF (e.g., [6S]-5-methyl-THF, or [6R,S]-5-methyl-THF) contains4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In some embodiments,administered 5-methyl-THF (e.g., [6S]-5-methyl-THF) is tetraglutamated5-methyl-THF. In some embodiments administered 5-methyl-THF (e.g.,[6S]-5-methyl-THF) is pentaglutamated 5-methyl-THF. In some embodiments,administered 5-methyl-THF (e.g., [6S]-5-methyl-THF) is hexaglutamated5-methyl-THF. In some embodiments, the 5-methyl-THF is encapsulated in aliposome. In some embodiments, (a) the 5-methyl-THF and oxaloplatin areencapsulated in the same liposome, (b) the 5-methyl-THF and 5-FU areencapsulated in the same liposome, (c) the oxaloplatin and 5-FU areencapsulated in the same liposome, or (d) the 5-methyl-THF, oxaloplatin,and 5-FU are encapsulated in the same liposome. In additionalembodiments, the 5-methyl-THF, oxaloplatin and 5-FU, are administered toa subject having or at risk of having cancer. In some embodiments, thecancer is selected from: colorectal cancer, breast cancer, gastriccancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lungcancer (e.g., non-small cell lung cancer and/or adenocarcinoma), headand neck cancer, ovarian cancer, gallbladder cancer, and basal cellcancer. In particular embodiments, the cancer is colorectal cancer. Infurther embodiments, 5-methyl-THF is administered in combination withoxaloplatin, 5-FU, and an antibody that binds VEGF (e.g., bevacizumab).

In some embodiments, polyglutamated 5-formyl-THF is administered incombination with a platinum-based agent (e.g., cisplatin, carboplatin,and oxaliplatin). In some embodiments, the administered 5-formyl-THF(e.g., [6S]-5-formyl-THF, or [6R,S]-5-formyl-THF) contains 4, 5, 2-10,4-6, or more than 5, glutamyl groups. In some embodiments, administered5-formyl-THF is tetraglutamated 5-formyl-THF. In some embodiments,administered 5-formyl-THF is pentaglutamated 5-formyl-THF. In someembodiments, administered 5-formyl-THF is hexaglutamated 5-formyl-THF.In some embodiments, polyglutamated [6S]-5-formyl-THF is administered incombination with a platinum-based agent. In some embodiments,polyglutamated [6R,S]-5-formyl-THF is administered in combination with aplatinum-based agent. In some embodiments, the 5-formyl-THF isadministered separately from the platinum-based agent. In someembodiments, the 5-formyl-THF is administered at the same time (e.g.,concurrently or serially) as the platinum-based agent. In someembodiments, the 5-formyl-THF and platinum-based agent are encapsulatedin the same liposome. In particular embodiments, the 5-formyl-THFcomposition is administered in combination with oxaliplatin. In someembodiments, the 5-formyl-THF is administered separately from theoxaloplatin. In some embodiments, the 5-formyl-THF is administered atthe same time (e.g., concurrently or serially) as oxaloplatin. In someembodiments, the 5-formyl-THF and oxaloplatin are encapsulated in thesame liposome.

In some embodiments, 5-formyl-THF is administered in combination with aplatinum-based agent (e.g., cisplatin, carboplatin, and oxaliplatin) anda pyrimidine analog. In some embodiments, the administered 5-formyl-THF(e.g., [6S]-5-formyl-THF, or [6R,S]-5-formyl-THF) contains 4, 5, 2-10,4-6, or more than 5, glutamyl groups. In some embodiments, administered5-formyl-THF (e.g., [6S]-5-formyl-THF) contains 4 glutamyl groups. Insome embodiments, administered 5-formyl-THF (e.g., [6S]-5-formyl-THF)contains 5 glutamyl groups. In some embodiments, administered5-formyl-THF (e.g., [6S]-5-formyl-THF) contains 6 glutamyl groups. Inadditional embodiments, the administered pyrimidine analog is selectedfrom: ancitabine, azacitidine, 6-azauridine, carmofur, cytosinearabinoside, dideoxyuridine, doxifluridine, enocitabine, floxuridine,and 5-FU. In some embodiments, polyglutamated [6S]-5-formyl-THF isadministered in combination with a platinum-based agent (e.g.,cisplatin, carboplatin, and oxaliplatin) and a pyrimidine analog. Insome embodiments, polyglutamated [6R,S]-5-formyl-THF is administered incombination with a platinum-based agent (e.g., cisplatin, carboplatin,and oxaliplatin) and a pyrimidine analog. In some embodiments, the5-formyl-THF is administered separately from the platinum-based agentand/or the pyrimidine analog. In some embodiments, the 5-formyl-THF isadministered at the same time (e.g., concurrently or serially) as theplatinum-based agent and/or the pyrimidine analog. In some embodiments,the 5-formyl-THF is encapsulated in a liposome. In some embodiments, (a)the 5-formyl-THF and platinum-based agent are encapsulated in the sameliposome, (b) the 5-formyl-THF and a pyrimidine analog are encapsulatedin the same liposome, (c) the platinum-based agent, and a pyrimidineanalog are encapsulated in the same liposome, or (d) the 5-formyl-THF,platinum-based agent, and a pyrimidine analog are encapsulated in thesame liposome.

In particular embodiments, 5-formyl-THF is administered in combinationwith oxaloplatin and 5-FU. In some embodiments, the administered5-formyl-THF (e.g., [6S]-5-formyl-THF, or [6R,S]-5-formyl-THF) contains4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In some embodiments,administered 5-formyl-THF (e.g., [6S]-5-formyl-THF) contains 4 glutamylgroups. In some embodiments, administered 5-formyl-THF (e.g.,[6S]-5-formyl-THF) contains 5 glutamyl groups. In some embodiments,administered 5-formyl-THF (e.g., [6S]-5-formyl-THF) contains 6 glutamylgroups. In some embodiments, the 5-formyl-THF is encapsulated in aliposome. In some embodiments, (a) the 5-formyl-THF and oxaloplatin areencapsulated in the same liposome, (b) the 5-formyl-THF and 5-FU areencapsulated in the same liposome, (c) the oxaloplatin and 5-FU areencapsulated in the same liposome, or (d) the 5-formyl-THF, oxaloplatin,and 5-FU are encapsulated in the same liposome. In additionalembodiments, the 5-formyl-THF, oxaloplatin and 5-FU, are administered toa subject having or at risk of having cancer. In some embodiments, thecancer is selected from: colorectal cancer, breast cancer, gastriccancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lungcancer (e.g., non-small cell lung cancer and/or adenocarcinoma), headand neck cancer, ovarian cancer, gallbladder, and basal cell cancer. Inparticular embodiments, the cancer is colorectal cancer. In furtherembodiments, 5-formyl-THF is administered in combination withoxaloplatin, 5-FU, and an antibody that binds VEGF (e.g., bevacizumab).

In additional embodiments, treatment methods provided herein compriseadministering a αPTHF composition in combination with an antibody thatbinds VEGF (e.g., bevacizumab). In some embodiments the disclosureprovides administering the αPTHF composition in combination with anantibody that binds VEGF and 5-FU. In some embodiments the disclosureprovides administering the αPTHF composition in combination with anantibody that binds VEGF and oxaloplatin. In further embodiments thedisclosure provides administering the αPTHF composition in combinationwith an antibody that binds VEGF, 5-FU, and oxaloplatin.

In additional embodiments, treatment methods provided herein compriseadministering a αPTHF composition in combination with an antibody thatbinds EGFR (e.g., panitumumab or cetuximab). In some embodiments thedisclosure provides administering the αPTHF composition in combinationan antibody that binds EGFR and 5-FU. In some embodiments the disclosureprovides administering the αPTHF composition in combination with anantibody that binds EGFR and oxaloplatin. In further embodiments thedisclosure provides administering the αPTHF composition in combinationwith an antibody that binds EGFR, 5-FU and oxaloplatin. In additionalembodiments, the liposome composition is administered to a subjecthaving or at risk of having colorectal cancer, breast cancer, gastriccancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lungcancer (e.g., non-small cell lung cancer and/or adenocarcinoma), headand neck cancer, ovarian cancer, gallbladder, or basal cell cancer. Infurther embodiments, the liposome composition is administered to asubject having or at risk of having colorectal cancer.

In particular embodiments, treatment methods provided herein compriseadministering a αPTHF composition described herein in combination with5-fluorouracil (5-FU) and irinotecan (FOLFIRI.) In some embodiments, theαPTHF composition is encapsulated in a liposome. In some embodiments,the αPTHF composition and 5-FU are encapsulated in a liposome. In someembodiments, the αPTHF composition and irinotecan are encapsulated in aliposome. In some embodiments, the αPTHF composition, 5-FU, andirinotecan are encapsulated in the same liposome. In additionalembodiments, the liposome composition is administered to a subjecthaving or at risk of having a cancer selected from: colorectal cancer,breast cancer, gastric cancer (e.g., stomach cancer), pancreatic cancer,liver cancer, lung cancer (e.g., non-small cell lung cancer and/oradenocarcinoma), head and neck cancer, ovarian cancer, gallbladder, orbasal cell cancer. In further embodiments, the liposome composition isadministered to a subject having or at risk of having colorectal cancer.In particular embodiments, the disclosure provides the administration ofa αPTHF composition described herein in combination with capecitabine.In some embodiments, the αPTHF composition is encapsulated in aliposome. In some embodiments, the administered αPTHF and capecitabineare encapsulated in the same liposome.

V. Kits Comprising αPTHF Compositions

The disclosure also provides kits that comprise the αPTHF compositionsdescribed herein and that can be used to perform the methods describedherein. In certain embodiments, a kit comprises at least one purifiedαPTHF composition in one or more containers.

In some embodiments the kits include a dosage amount (e.g., as used fortherapy or diagnosis) of at least one αPTHF compositions (e.g., a αPTHFliposome), or pharmaceutical formulation thereof, as disclosed herein.Kits may further comprise suitable packaging and/or instructions for useof the composition. Kits may also comprise a means for the delivery forthe composition, or pharmaceutical formulation thereof, such as asyringe for injection or other device as described herein and known tothose of skill in the art. One of skill in the art will readilyrecognize that the disclosed αPTHF compositions can be readilyincorporated into one of the established kit formats which are wellknown in the art.

Further provided are kits that comprise a αPTHF compositions as well asat least one additional therapeutic agent. In certain embodiments, thesecond (or more) therapeutic agent is an anti-metabolite. In certainembodiments, the second (or more) therapeutic agent is achemotherapeutic agent.

The following examples are intended to illustrate but not to limit thedisclosure in any manner, shape, or form, either explicitly orimplicitly. While they are typical of those that might be used, otherprocedures, methodologies, or techniques known to those skilled in theart may alternatively be used without departing from the scope of thepresent disclosure.

FIG. 1B-1N shows chemical formulae of exemplary polyglutamated alphatetrahydrofolate compositions encompassed by the disclosure.

EXAMPLES Example 1: Liposomal Gamma Polyglutamated PemetrexedCompositions

Methods:

Production of Gamma Hexaglutamated Pemetrexed (γHgTHF) Liposomes

Briefly Gamma Hexaglutamated Pemetrexed (gGM6) and D alphahexaglutamated Pemetrexed (gDGM6) was encapsulated in liposomes by thefollowing procedure. First, the lipid components of the liposomemembrane were weighed out and combined as a concentrated solution inethanol at a temperature of around 65° C. In this example, the lipidsused were hydrogenated soy phosphatidylcholine, cholesterol, andDSPE-PEG-2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (poly-ethyleneglycol)-2000]). The molar ratio of HSPC:Cholesterol:PEG-DSPE wasapproximately 3:2:0.15. Next, the gGM6 or gDGM6 was dissolved in 5%dextrose at a concentration of 100-150 mg/ml with a pH of 6.5-6.9. Thedrug solution was heated up to 65° C. The ethanolic lipid solution wasinjected into the gGM6 or gDGM6 solution using a small-bore needle.During this step the drug solution was well stirred using a magneticstirrer. The mixing was performed at an elevated temperature (63° C.-72°C.) to ensure that the lipids were in the liquid crystalline state (asopposed to the gel state that they attain at temperatures below thelipid transition temperature Tm=51° C.-54° C.). As a result, the lipidswere hydrated and form multiple bilayer (multilamellar) vesicles (MTHF)containing gGM6 or gDGM6 in the aqueous core.

Downsizing of MTHF's Using Filter Extrusion

The MTHFs were fragmented into unilamellar (single bilayer) vesicles ofthe desired size by high-pressure extrusion using three passes throughstacked (track-etched polycarbonate) membranes. The first pass wasperformed through stacked membranes consisting of two layers with a poresize of 200 nm. The remaining two passes were through the stackedmembranes consisting of three layers with a pore size of 100 nm. Duringextrusion, the temperature was maintained above the Tm to ensureplasticity of the lipid membranes. As a result of the extrusion, largeand heterogeneous in size and lamellarity MTHFs turned into small,homogenous (90-125 nm) unilamellar vesicles (ULV) that sequestered thedrug in their interior. A Malvern Zetasizer Nano ZS instrument(Southborough, Mass.) with back scattering detector (90°) was used formeasuring the hydrodynamic size (diameter) at 25° C. in a quartz microcuvette. The samples were diluted 50-fold in formulation matrix beforeanalysis.

Purification of Liposomes

After the ULV's containing gGM6 or gDGM6 had been produced, theextra-liposomal free drug was removed using columns for small volume ortangential flow diafiltration against a suitable buffer for largevolume. Although any buffer solution can be used, in this example thebuffer used was 5 mM HEPES, 145 mM Sodium Chloride, pH 6.7. Uponcompletion of purification, filter sterilization was performed using a0.22 micron filter.

Production of Alpha Hexaglutamated Pemetrexed (αHgTHF) Liposomes

Briefly L alpha hexaglutamated pemetrexed (aG6) and D alphahexaglutamated pemetrexed (aDG6) were encapsulated in liposomes by thefollowing procedure. First, the lipid components of the liposomemembrane were weighed out and combined as a concentrated solution inethanol at a temperature of around 65° C. In this example, the lipidsused were hydrogenated soy phosphatidylcholine, cholesterol, andDSPE-PEG-2000(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly-ethylene glycol)-2000]). The molar ratio ofHSPC:Cholesterol:PEG-DSPE was approximately 3:2:0.15. Next, the aG6 oraDG6 was dissolved in 5% dextrose at a concentration of 150 mg/ml with apH of 6.5-6.9. The drug solution was heated up to 65° C. The ethanoliclipid solution was injected into the aG6 or aDG6 solution using asmall-bore needle. During this step the drug solution was well stirredusing a magnetic stirrer. The mixing was performed at an elevatedtemperature (63° C.-72° C.) to ensure that the lipids were in the liquidcrystalline state (as opposed to the gel state that they attain attemperatures below the lipid transition temperature Tm=51° C.-54° C.).As a result, the lipids were hydrated and form multiple bilayer(multilamellar) vesicles (MTHF) containing aG6 or aDG6 in the aqueouscore.

Downsizing of MTHF's Using Filter Extrusion

The MTHFs were fragmented into unilamellar (single bilayer) vesicles ofthe desired size by high-pressure extrusion using three passes throughstacked (track-etched polycarbonate) membranes. The first pass wasperformed through stacked membranes consisting of two layers with a poresize of 200 nm. The remaining two passes were through the stackedmembranes consisting of three layers with a pore size of 100 nm. Duringextrusion, the temperature was maintained above the Tm to ensureplasticity of the lipid membranes. As a result of the extrusion, largeand heterogeneous in size and lamellarity MTHFs turned into small,homogenous (90-125 nm) unilamellar vesicles (ULV) that sequestered thedrug in their interior. A Malvern Zetasizer Nano ZS instrument(Southborough, Mass.) with back scattering detector (90°) was used formeasuring the hydrodynamic size (diameter) at 25° C. in a quartz microcuvette. The samples were diluted 50-fold in formulation matrix beforeanalysis.

Purification of Liposomes

After the ULV's containing aG6 or aDG6 had been produced, theextra-liposomal gG6 was removed using columns for small volume ortangential flow diafiltration against a suitable buffer for largevolume. Although any buffer solution can be used, in this example thebuffer used was 5 mM HEPES, 145 mM Sodium Chloride, pH 6.7. Uponcompletion of purification, filter sterilization was performed using a0.22 micron filter. The typical characteristics of liposomal derivativesare shown in the table below.

Starting Encapsulation Final Drug/Lipid Zeta con. efficiency con. RatioDiameter PDI potential Lps 1 mg/ml 4.75% 0.031 mg/ml 25-30 g/mol 122.8nm 0.021 −1.14 mV aDG6 Lps 1 mg/ml 5.90% 0.039 mg/ml 25-30 g/mol 100.2nm 0.018 −1.90 mV aG6 LpS 150 mg/ml  36% 8.0 mg/ml 230-260 g/mol 104 nm0.04 −2.73 mV aG6

Dose Response Study of Alpha HGP (Hexaglutamated Pemetrexed) andLiposomes

A dose response study was performed using liposomes containinghexaglutamated pemetrexed that were prepared essential as describedabove.

Cell viability was determined by CellTiter-Glo® (CTG) luminescent cellviability assay on Day 3 (48 hour) and Day 4 (72 hour). This assaydetermines the number of viable cells in culture based on quantifyingATP that is present within, which in turn signals the presence ofmetabolically active cells. The CTG assay uses luciferase as a readout.To assess cell viability Dose response inhibition of pemetrexed, HGP andliposomes on different cancer cell growth were investigated usingCellTiter-Glo® luminescent cell viability assay. Human cancer cells wereharvested, counted and plated at a same cell density on Day 0. A seriesof 8 dilutions of each test article were added to the cells on Day 1.Dose response curve were generated and fit using GraphPad Prism and IC50of each test article were calculated. A lower the IC50 is, the morepotent the test article is in term of cancer cell growth inhibition.

Cells were seeded into 96-well plate at a cell density of 5×10⁴ cellsper well in 100 μl of fresh media on Day 0. Eight serial 2-folddilutions of each test article in culture medium were generated andadded to cells in triplicate on Day 1. In addition, three wells of cellswere treated with vehicle (HBS for free drug or empty liposome forliposomal HGP) alone as a control.

On Days 3 and 4, 100 μl of CellTiterGlo® Reagent were added to each welland incubated at room temperature for 15 minutes. Luciferaseluminescence were recorded for each well. In addition, 8 serial 2-folddilutions of the vehicle (HBS or empty liposome) in culture medium wereadded into empty wells and included in the assay to generate thebackground luminescence signals. Luciferase signals were normalized bysubtracting the background luminescence signal out of the read-outsrespectively.

Human Normal Primary Bone Marrow CD34+ Cells were obtained from ATCC.(ATCC Catalog Number PCS-800-012). Cells were thawed at 37° C. for 1minute and then placed on ice. The cells were then resuspended inStemSpan SFEM (Stem Cell Tech Catalog Number 9650) plus 10% heatinactivated fetal bovine serum (Corning 35-015-CV). The cells wereplated into 96 well culture plates at a density of 2.5×10⁴ cells/well.The following day, live cells were collected via centrifugation andresuspended in neutrophil growth media (StemSpan SFEM plus 10% HeatInactivated fetal bovine serum plus 100 ng/ml human stem cell factor(Sigma Catalog Number H8416), 20 ng/ml human granulocytecolony-stimulation factor (Sigma Catalog Number H5541), and 10 ng/mlhuman recombinant IL3 (Sigma SRP3090) at a density of 2.5×104cells/well. Cells were incubated at 37° C. for 10 days. Fresh media wasadded every two days. Mature neutrophils were then collected and platedin 96 well plates at a density of 1×10⁴ cells/well and incubated at 37°C. overnight. The next day, test article or vehicle was resuspended inneutrophil growth media and added to the plates. The cells were thenincubated for either 48 hours or 72 hours at 37° C. and then assayed ateach time point using the Cell Titer Glo Assay (Promega Catalog #G7572).

Methodologies used for cell line AML12 (non-cancerous liver cells) andCCD841 (non-cancerous colon epithelial cells) are similar to the methodsused for cancer cells.

Results

In a set of dose response experiments, 6 cell lines representingdifferent types of cancers, namely HT-29 (colon cancer), H2342 (NSCLC,adenocarcinoma subtype), H292 (NSCLC, adenocarcinoma subtype), SW620(CRC), H1806 (triple negative breast cancer) and OAW28 (ovarian cancer),were studied (FIG. 2). Treatment consisted of exposure for 48 hoursusing 2 different encapsulated derivatives of liposomal alpha pemetrexedhexaglutamate, namely liposomal alpha L hexaglutamate (liposomal aG6)and its mirror image, liposomal alpha D hexaglutamate (liposomal aDG6)also referred to as its corresponding enantiomer.

The relative potency of the above mentioned derivatives as compared topemetrexed, following exposure over 48 hours, is represented in FIG. 2.The relative potency of treatment using the various derivatives, asshown in this figure was calculated by dividing the IC50 of pemetrexedby the IC50 of the liposomal alpha pemetrexed hexaglutamate for eachcell line. As shown in this figure, in all cell lines, the potency ofliposomal alpha pemetrexed hexaglutamate well exceeded that ofpemetrexed. By way of example, consider the NSCLC cell line H292. Asshown in the figure, the potency of liposomal alpha pemetrexedhexaglutamate was ≥50-fold that of pemetrexed. This suggests that a 2%or lower dose of the liposomal alpha pemetrexed hexaglutamate could havethe same treatment effect as a 100% dose of pemetrexed.

As stated in some instances increased uptake of payload can be achievedby targeting the liposomal delivery vehicle using antibody such asFolate Receptor Alpha. By way of example in the next two experimentsLiposomal L Gamma G6/Lps Hexa gG6 was encapsulated using the methodspreviously described above. Subsequently, pemetrexed, liposomal gammapemetrexed hexaglutamate derivatives (Liposomal L gamma G6/Lps Hexa gG6)and Folate Receptor Alpha Targeted Liposomal L Gamma G6 (LiposomalgG6-FR1Ab), Free (unencapsulated) L gamma G6 were tested for cytotoxicactivity on representative cell lines in non small cell lung cancercells (NCI-H2342) and colorectal cancer cells (HT-29) as shown in FIG. 3and FIG. 4 respectively. These data show that both liposomal L gammapemetrexed hexaglutamate and Folate Receptor Alpha Targeting liposomal Lgamma pemetrexed hexaglutamate are more potent than pemetrexed in bothcell lines. In general Folate Receptor Alpha Antibody targetingliposomes show the highest potency. By contrast free L gamma G6 has thelowest potency due to its inability to traffic across cell membraneseffectively.

Cancer cell viability studies comparing the liposomal alpha pemetrexedhexaglutamate derivatives (liposomal L alphaG6/Lps Hexa aG6 andliposomal D alphaG6/Lps Hexa aDG6) and pemetrexed for cytotoxic activityon representative cell lines in breast, lung and ovarian cancer areshown in FIGS. 5-7. These data show that both liposomal alpha Lpemetrexed hexaglutamate and liposomal alpha D pemetrexed hexaglutamateare more potent than pemetrexed. Further, as an indicator of efficacy,the results of the experiments on the same cell lines depicted atvarious dose levels ranging from 16 to 128 nM in FIGS. 8-10. As shown inthese figures, at each of these dose ranges, liposomal alpha Lpemetrexed hexaglutamate and liposomal alpha D pemetrexed hexaglutamateare superior to pemetrexed in terms of inhibiting cancer cells for thelung and breast cancer cell lines. In the ovarian cancer cell line,pemetrexed at the dose of 128 nM, appears to be equally effective asliposomal alpha pemetrexed hexaglutamate, whereas the liposomal alphapemetrexed hexaglutamate at the dose of 32 nM and 64 nM has a bettertreatment effect than pemetrexed; at 16 nM the treatment effect is lowerand similar in magnitude for liposomal alpha pemetrexed hexaglutamateand pemetrexed.

The major toxicities seen in patients treated with pemetrexed is bonemarrow suppression which manifests as a decrease in blood countsincluding neutrophil counts (a type of white blood cells). There is alsosome adverse effect on the lining of the mouth and gut that manifests asdiarrhea and mucositis, as well as an adverse effect on the liver insome instances. To assess the above-mentioned toxicities, treatment ofthe liposomal alpha pemetrexed hexaglutamate derivatives (L and D) andpemetrexed was measured at 48 hours on CD34+ cells that weredifferentiated into neutrophils, CCD841 colon epithelium cells and AML12liver cells. As shown in FIG. 11, liposomal alpha pemetrexedhexaglutamate is significantly less toxic to differentiating humanneutrophils in contrast to pemetrexed. This is also supported byneutrophil counts that are better preserved following treatment with theliposomal alpha L pemetrexed hexaglutamate or liposomal alpha Dpemetrexed hexaglutamate compared to pemetrexed, at dose ranges from 16nM to 128 nM (FIG. 12). Strikingly, there does not appear to be anytoxicity to the liver cells following treatment with liposomal L alphapemetrexed hexaglutamate or liposomal alpha D pemetrexed hexaglutamateat the dose levels studied (FIG. 13). In contrast, pemetrexed at alldoses studied is leading to a reduction in the liver cell counts ofapproximately 40%. And finally, the same trend is seen followingtreatment of epithelial colon cells (FIG. 14). As shown in this figure,pemetrexed at all doses studied is leading to approximately a ≥50%decrease in the number of cells compared to approximately a 20% or lessdecrease after treatment with liposomal alpha L pemetrexed hexaglutamateand liposomal alpha D pemetrexed hexaglutamate.

Example 2: Polyglutamated Antifolate-Cisplatin Complexes (PGPD) Methods:

Folate Analogues also known as antifolate have been an importantanticancer treatment for the last 70 years. Used in this setting thisclass of anti-cancer drugs interferes with various enzymes in theimportant folate metabolic pathway. This can result in impairedpyrimidine and purine (DNA and RNA) synthesis, impaired amino acidglycine and serine metabolism, impaired redox response and impairedmethylation processes within the cell.

In in clinical practice, antifolates such as pemetrexed andtetrahydrofolate are often used in combination with platinum agents suchas cisplatin and carboplatin. The combinations result in enhancedefficacy. In this context, we set out to coencapsulated thepolyglutamates with platinum agents in a specific ratio to facilitatecontrolled delivery of a predetermined ratio of the two anticancer drugsnamely a polyglutamated antifolate and a platinum analogue. Wesurprisingly discovered that long forms of polyglutamate antifolate(e.g., pentaglutamated antifolate) forms a complex with cisplatin thatis stable at high pH, and that this complex disassociates intopolyglutamate and cisplatin at low pH. Low pH is believed to be occur inmany tumor cells and the tumor cell environment, particularly in hypoxicsettings. Application of this discovery provides the ability tofacilitate the delivery of combinations of alpha polyglutamatedpemtrexed (αPPMX) and therapeutic agents such as cisplatin to targetcells such as tumor cells and to release the drugs from the complex inphysiologically relevant low pH conditions.

Production of Polyglutamated Antifolates-DDAP (Cisplatin) Complexes(PGPD)

To produce (Polyglutamated antifolates-cisplatin DDAP Complex), alphahexaglutamate (aG6) and Diammine dicarboxylic acid platinum (DDAP) wasused. The process of complexation was dependent on the presence ofChlorinated platinum compound and pH conditions. The complexation wasachieved by a nucleophilic attack on one or two carboxyl groups ofglutamate by the platinate derivative. Briefly the complex was formed bythe following procedure. First, the active compound DDAP was weighed anddissolved in 5% dextrose. After the DDAP dissolution step, aG6 wasweighed out and added to the DDAP-Captisol® (solution and allowed tostir for 1 hour at 45-55° C. The pH of the solution was adjusted to6.5-7.0 using 1N NaOH and the solution was stirred for 1-2 hour. Theformation of complex was confirmed visually. However when the pH isadjusted to acidic pH of 3-5, the color reverted back to its original,indicating the decomplexatoin of the polyglutamated antifolate andcisplatin. FIG. 15 depicts a schematic providing possible scenariosexplaining the observed pH dependent complex formation between thepolyglutamated antifolate and cisplatin.

Complex formation was confirmed using HPLC which showed two distinctpeaks that merge into 1 large peak at high pH of 6.5 to 7.5 and thenreappear at low pH of 3-5. Repeating the experiment without Captisolshowed that complex formation was independent of Captisol®.

Production of Pentaglutamated Pemetrexed-DDAP Complex (PGPD) Liposomes

Briefly PGPD was encapsulated in liposomes by the following procedure.First, the lipid components of the liposome membrane was weighed out andcombined as a concentrated solution in ethanol at a temperature ofaround 65° C. In this example, the lipids used were hydrogenated soyphosphatidylcholine, cholesterol, and DSPE-PEG-2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]). The molar ratio of HSPC:Cholesterol:PEG-DSPE wasapproximately 3:2:0.15. Next, PGPD was prepared as described above. ThePGPD drug solution was heated up to 65° C. The ethanolic lipid solutionwas injected into the PGPD solution using a small-bore needle. Duringthis step the drug solution was well stirred using a magnetic stirrer.The mixing was performed at an elevated temperature (63° C.-72° C.) toensure that the lipids were in the liquid crystalline state (as opposedto the gel state that they attain at temperatures below the lipidtransition temperature Tm=51° C.-54° C.). As a result, the lipids werehydrated and formed multiple bilayer (multilamellar) vesicles (MTHF)containing PGPD in the aqueous core.

Downsizing of MTHF's Using Filter Extrusion

The MTHFs were fragmented into unilamellar (single bilayer) vesicles ofthe desired size by high-pressure extrusion using two passes throughstacked (track-etched polycarbonate) membranes. The stacked membraneshave two layers with a pore size of 200 nm and six layers with a poresize of 100 nm. During extrusion, the temperature was maintained abovethe Tm to ensure plasticity of the lipid membranes. Because of theextrusion, large and heterogeneous in size and lamellarity MTHFs turninto small, homogenous (100-120 nm) unilamellar vesicles (ULV) thatsequester the drug in their interior. A Malvern Zetasizer Nano ZSinstrument (Southborough, Mass.) with back scattering detector (90°) wasused for measuring the hydrodynamic size (diameter) at 25° C. in aquartz micro cuvette. The samples were diluted 50-fold in formulationmatrix before analysis.

Purification of Liposomes:

After the ULV's containing PGPD had been produced, the extra-liposomalPGPD was removed using columns for small volume or tangential flowdiafiltration against a suitable buffer for large volume. Although manydifferent buffers known in the art could have been used, in this examplethe buffer used was 5 mM HEPES, 145 mM Sodium Chloride, pH 6.7. Uponcompletion of purification, filter sterilization was performed using a0.22-micron filter. The liposomes prepared according to the aboveprocedures were determined to have a diameter of 116.6 nm, a PDI of0.083, and a zeta potentials of −2.05 mV.

Example 3: Targeted Liposome Polyglutamated Pemetrexed Cell DeliveryMethods: Production of Targeted Gamma Hexaglutamated Pemetrexed (HGP)Liposomes

Gamma HGP (gG6) was encapsulated in liposomes and the liposomes weredownsized and purified according to procedures essentially as set forthabove in Example 1.

Antibody Conjugation

Activated liposomes were prepared by adding DSPE-PEG-maleimide to thelipid composition. The liposomes contain four different lipids:hydrogenated soy phosphatidylcholine (HSPC), cholesterol,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG-2000), and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG-maleimide), in ratios of3:2:0.1125:0.0375.

Antibody thiolation was accomplished through use of Traut's reagent(2-iminothiolane) to attach a sulfhydryl group onto primary amines.Antibody was suspended in PBS at a concentration of 0.9-1.6 mg/ml.Traut's reagent (14 mM) was added to antibody solution at a finalconcentration of 1-5 mM and then removed through dialysis after one-hourincubation at room temperature. Thiolated antibody was added toactivated liposome at a ratio of 60 g/mol phosphate lipids, and thereaction mixture was incubated for one hour at room temperature andover-night at 4 uL-cysteine was used to terminate the reaction andunconjugated antibody was removed through dialysis.

Exemplary Direct and Post Insertion Antibody-Liposome Conjuation Methodsare Provided Below. Exemplary Antibody Conjugation Method 1: DirectConjugation

Antibody or its fragments, such as Fab or scFv, can be conjugateddirectly onto thiol-reactive liposome. Thiol-reactive liposomes areprepared by adding DSPE-PEG-maleimide to the lipid composition. Theliposomes contain four different lipids: hydrogenated soyphosphatidylcholine (HSPC), cholesterol,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG-2000), and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG-maleimide), in ratios of3:2:0.1125:0.0375.

Antibody (or its fragments, such as Fab or scFv) thiolation isaccomplished through use of Traut's reagent (2-iminothiolane) to attacha sulfhydryl group onto primary amines. Antibody (or its fragment) issuspended in PBS at a concentration of 0.9-1.6 mg/ml. Traut's reagent(14 mM) is added to antibody (or its fragment) solution at a finalconcentration of 1-5 mM and then removed through dialysis after one-hourincubation at room temperature. Thiolated antibody (or its fragment) isadded to thiol-reactive liposome at a ratio of 60 g/mol phosphatelipids, and the reaction mixture is incubated for one hour at roomtemperature and over-night at 4° C. L-cysteine is used to terminate thereaction and unconjugated antibody (or its fragment) is removed throughdialysis.

Antibody or its fragments, such as Fab or scFv, which contains acysteine residue at the C-terminal can be conjugated directly onto theliposome by incubating a reduced antibody (or its fragment) withthiol-reactive liposome. Antibody (or its fragment) with a cysteine tailis dissolved and reduced by a 10-20 mM reducing reagent (such as2-mercaptoethylamine, cysteine, or dithioerythritol) at pH<7. The excessreducing reagent is removed thoroughly by size exclusion chromatographyor dialysis. The purified and reduced antibody (or its fragment) can bedirectly conjugated to the thiol-reactive liposome.

Exemplary Antibody Conjugation Method 2: Post Insertion

Antibody or its fragments, such as Fab or scFv, which contains acysteine residue at the C-terminal can be conjugated and incorporatedinto the liposome through a “post insertion” method. Micelles ofthiol-reactive lipopolymer (such as DSPE-PEG-maleimide) is prepared bydissolving in an aqueous solution at 10 mg/ml. Antibody (or itsfragment) with a cysteine tail is dissolved and reduced by a 10-20 mMreducing reagent (such as 2-mercaptoethylamine, cysteine, ordithioerythritol) at pH<7. The excess reducing reagent is removedthoroughly by size exclusion chromatography or dialysis. The purifiedand reduced antibody (or its fragment) is then incubated with themicelles of thiol-reactive lipopolymers at a molar ratio of 1:4. At theend of the reaction, the excess maleimide groups are quenched by a smallamount of cysteine (1 mM) or mercaptoethanol. Unconjugated antibody (orits fragment) is removed by size exclusion chromatography. Purifiedconjugated micelles is then incubated with liposome at 37° C. orelevated temperature.

Physical Characteristics of the Nanoparticles

Starting Encapsulation Final Drug/Lipid Zeta con. efficiency con. RatioDiameter PDI potential Lps 20 mg/ml 10.60% 1.39 mg/ml 35-50 g/mM lipids114.9 nm 0.035 −1.76 mV gG6

Dose Response Study of HGP (Pentaglutamated Pemetrexed) and Liposomes.

Cell viability was determined by CellTiter-Glo® (CTG) luminescent cellviability assay on Day 3 (48 hour) and Day 4 (72 hour). This assaydetermines the number of viable cells in culture based on quantifyingATP that was present within, which in turn signals the presence ofmetabolically active cells. The CTG assay uses luciferase as a readout.To assess cell viability Dose response inhibition of pemetrexed, HGP andliposomes on different cancer cell growth were investigated usingCellTiter-Glo® luminescent cell viability assay. Human cancer cells wereharvested, counted and plated at a same cell density on Day 0. A seriesof 8 dilutions of each test article were added to the cells on Day 1.Dose response curve were generated and fit using GraphPad Prism and IC50of each test article were calculated. A lower the IC50 is, the morepotent the test article was in term of cancer cell growth inhibition.

Cells were seeded into 96-well plate at a cell density of 5×10⁴ cellsper well in 100 μl of fresh media on Day 0. Eight serial 2-folddilutions of each test article in culture medium were generated andadded to cells in triplicate on Day 1. In addition, three wells of cellswere treated with vehicle (HBS for free drug or empty liposome forliposomal HGP) alone as a control.

On Days 3 and 4, 100 μl of CellTiterGlo® Reagent were added to each welland incubated at room temperature for 15 minutes. Luciferaseluminescence were recorded for each well. In addition, 8 serial 2-folddilutions of the vehicle (HBS or empty liposome) in culture medium wereadded into empty wells and included in the assay to generate thebackground luminescence signals. Luciferase signals were normalized bysubtracting the background luminescence signal out of the read-outsrespectively.

Human Normal Primary Bone Marrow CD34+ Cells were obtained from ATCC.(ATCC Catalog Number PCS-800-012). Cells were thawed at 37° C. for 1minute and then placed on ice. The cells were then resuspended inStemSpan SFEM (Stem Cell Tech Catalog Number 9650) plus 10% heatinactivated fetal bovine serum (Corning 35-015-CV). The cells wereplated into 96 well culture plates at a density of 2.5×10⁴ cells/well.The following day, live cells were collected via centrifugation andresuspended in neutrophil growth media (StemSpan SFEM plus 10% HeatInactivated fetal bovine serum plus 100 ng/ml human stem cell factor(Sigma Catalog Number H8416), 20 ng/ml human granulocytecolony-stimulation factor (Sigma Catalog Number H5541), and 10 ng/mlhuman recombinant IL3 (Sigma SRP3090) at a density of 2.5×10⁴cells/well. Cells were incubated at 37° C. for 10 days. Fresh media wasadded every two days. Mature neutrophils were then collected and platedin 96 well plates at a density of 1×10⁴ cells/well and incubated at 37°C. overnight. The next day, test article or vehicle was resuspended inneutrophil growth media and added to the plates. The cells were thenincubated for either 48 hours or 72 hours at 37° C. and then assayed ateach time point using the Cell Titer Glo Assay (Promega Catalog #G7572).

Methodologies used for cell line AML12 (non-cancerous liver cells) andCCD841 (non-cancerous colon epithelial cells) are similar to the methodsused for cancer cells.

Results:

The dose response relationship of free pemetrexed gamma hexaglutamate(gG6), (non-targeted) liposomal gamma hexaglutamate (liposomal gG6),pemetrexed and folate receptor alpha targeting antibody (FR1Ab)liposomal pemetrexed gamma hexaglutamate (liposomal gG6-FR1Ab), in theNCI H2342 non-small cell lung cancer (NSCLC), adenocarcinoma subtype isshown in FIG. 3. The output is percentage of viable cells after 48 hoursof treatment as measured by luciferase luminescence. As shown in thisFIG. 3, the free pemetrexed gG6 appears to be the least potent asmeasured by IC50. Both the liposomal pemetrexed gG6 and the liposomalpemetrexed gG6-FR1Ab are 7-fold and 40-fold more potent, respectively,than free pemetrexed.

Similar data is shown for the HT-29 colon cancer cell line in FIG. 4that depict cell viability expressed as a percentage. As shown in thisfigure, free pemetrexed gG6 appears to be the least potent. In thisinstance, the liposomal pemetrexed gG6 is twice as potent as pemetrexedand the liposomal pemetrexed gG6-FR1Ab is 5-fold more potent than freepemetrexed.

Example 4: In Vivo Studies Methods: Safety Studies in Mice

Because some of the major toxicities associated with a pemetrexed basedtreatment are hematologic and hepatic, it is important to evaluate theeffect of Liposomal alpha G6 (Lp-aG6) in an in-vivo (murine) model andcompare the changes in hematologic and the liver serum chemistry panelfollowing treatment. To obtain this data an initial dose ranging studywas conducted using healthy female BALB/c mice (6-8 weeks old) whichwere purchased from The Jackson Laboratory (Bar Harbor, Me.). Prior tothe study, animals were weighed, randomized by weight, observed forclinical abnormalities, and distributed into groups (5 mice per group).Doses from 10 mg/kg up to 200 mg/kg were investigated to identify atolerable dose in mice. Treatments were administrated intravenously oncea week for four weeks. Body weight and detailed clinical observationwere recorded daily. At the end of study, Day 28, mice were euthanized,and blood and tissue were harvested from untreated Control mice and forthe mice treated with Liposomal aG6 40 mg/kg and Liposomal aG6 80 mg/kg.Whole blood was collected into K2-EDTA anticoagulant tubes forcomprehensive complete blood count (CBC) and serum was isolated forcomprehensive chemistry and was sent to IDEXX (Westbrook, Me.) on theday of collection.

Results:

In general, treatment with once weekly liposomal aG6 at two dose levelsof 40 mg/kg and 80 mg/kg for 4 weeks was well tolerated and there wereno major differences in weight compared to untreated controls. To assesssome of the effects on hematologic parameters, white blood cell (WBC)counts, neutrophil counts as well as platelet counts were measured aftertreatment with liposomal aG6 at two dose levels of 40 mg/kg and 80 mg/kgboth given once weekly for 4 weeks. As can be seen in FIG. 16, therewere no appreciable decreases in mean neutrophil, mean white blood celland mean platelet counts, after four weeks of treatment with LiposomalaG6 in treated animals compared to untreated control animals. Hemoglobinand reticulocyte indices were measured to assess the impact on red bloodcell. As shown in FIG. 17, there was a minimal decrease in meanhemoglobin concentrations at the higher dose level. In parallel there isa slight increase in mean reticulocytosis indices which suggests a bonemarrow's response to treatment by increasing red blood cell production.Altogether this effect seems minor as the mice hemoglobin levels aremaintained after 4 weeks of treatment. Taken together these data suggestthat at these dose levels, 40 mg/kg and 80 mg/kg once-weekly, there islittle impact on the bone marrow and related hematologic indices.

Another concern with pemetrexed is hepatic toxicity that has beenobserved in some patients treated with pemetrexed based therapy. Toassess hepatic well being in mice serum chemistries including serumaspartate transaminase (AST) and serum alanine transaminase (ALT) alongwith serum albumin were measured. As shown in FIG. 18, there were noappreciable increases in liver transaminases mean AST and mean ALTlevels at 4 weeks following treatment with Liposomal aG6 at the two doselevels of 40 mg/kg and 80 mg/kg both given once weekly for 4 weeks whencompared to untreated controls. There was no change in mean albuminlevels either. Taken together these data suggest a favorable safetyprofile for Liposomal aG6.

Preliminary Pilot Efficacy Study in Mice Xenografts

To assess whether there was any tumor control following treatment withliposomal alpha pemetrexed G6 (Lp-aG6) the pilot study was conducted. Inthis study Immunodeficient female Nude mice (Nu/J; 6-8 weeks old) werepurchased from The Jackson Laboratory (Bar Harbor, Me.). NCI-H292(Non-Small Cell Lung Cancer) cells were cultured in RPMI mediasupplemented with 10% Fetal Bovine Serum in a 37° C., 5% CO₂ incubator.1×10⁶ cells were inoculated subcutaneously into the dorsal hind flank ofeach mouse. Tumor volume and body weight were monitored twice everyweek. Tumor-bearing mice were randomized by tumor volume on Day 0 anddistributed into groups (5 mice per group): Control, Pemetexed, andLiposomal aG6. Pemetrexed was given intravenously at 167 mg/kg onceevery three weeks. This murine dose of 167 mg/kg every three weeks isequivalent to the FDA/EMA approved human dose and schedule of 500 mg/m²every three weeks. Liposomal aG6 was dosed intravenously at 80 mg/kgonce a week for four weeks. Tumor size was measured with a caliper andtumor burden is calculated using the following equations: tumorvolume=0.5× (tumor length)×(tumor width); Relative tumor volume=(tumorvolume/tumor volume on Day 0)×100%. This study is still ongoing butpreliminary data are shown in FIG. 19. In this figure, relative tumorvolume is displayed following treatment with Liposomal aG6 andpemetrexed. As can be seen from these preliminary data, liposomal aG6provides better tumor control when compared to pemetrexed.

Further Embodiments

In a non-limiting embodiment, of this disclosure, there is provided acomposition comprising alpha polyglutamated tetrahydrofolate.

In the composition of the immediately preceding paragraph, thecomposition may comprise pentaglutamated or hexaglutamatedtetrahydrofolate.

In the composition of any of the preceding two paragraphs, thecomposition may comprise alpha polyglutamated tetrahydrofolate which mayinclude pentaglutamated or hexaglutamated tetrahydrofolate.

A non-limiting example liposomal alpha polyglutamated tetrahydrofolate(L-αPTHF) composition may comprise a composition of any of the precedingthree paragraphs and the liposome may be optionally pegylated(PL-αPTHF).

In the L-αPTHF or PL-αPTHF composition of the immediately precedingparagraph, the alpha polyglutamated tetrahydrofolate may includepentaglutamated or hexaglutamated tetrahydrofolate.

In the L-αPTHF or PL-αPTHF composition of any of the preceding twoparagraphs, the liposome may be anionic or neutral.

In the L-αPTHF or PL-αPTHF composition of any of the preceding threeparagraphs, a targeting moiety may be attached to one or both of a PEGand the exterior of the liposome, and the targeting moiety may have aspecific affinity for a surface antigen on a target cell of interest(TL-αPTHF or TPL-αPTHF).

In the L-αPTHF or PL-αPTHF composition of any of the preceding fourparagraphs, a targeting moiety may be attached to one or both of a PEGand the exterior of the liposome and may be a polypeptide.

In the L-αPTHF or PL-αPTHF composition of any of the preceding fiveparagraphs, a targeting moiety may be attached to one or both a PEG andthe exterior of the liposome and may be an antibody or a fragment of anantibody.

In the L-αPTHF or PL-αPTHF composition of any of the preceding sixparagraphs, one or more of an immunostimulatory agent, a detectablemarker and a maleimide may be disposed on at least one of a PEG and theexterior of the liposome.

In the L-αPTHF or PL-αPTHF composition of any of the preceding sevenparagraphs, a polypeptide may bind an antigen with an equilibriumdissociation constant (Kd) in a range of 0.5×10⁻¹⁰ to 10×10⁻⁶ asdetermined using BIACORE® analysis.

In the L-αPTHF or PL-αPTHF composition of any of the preceding eightparagraphs, a polypeptide may specifically bind one or more folatereceptors selected from the group consisting of: folate receptor alpha(FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ).

A non-limiting exemplary method of killing a hyperproliferative cellthat includes contacting a hyperproliferative cell with a liposomalalpha polyglutamated tetrahydrofolate composition of any of thepreceding nine paragraphs.

In the method of the immediately preceding paragraph, thehyperproliferative cell is a cancer cell.

A non-limiting example method for treating cancer comprisesadministering an effective amount of the alpha polyglutamatedtetrahydrofolate composition of any of preceding paragraphs frompreceding paragraph eleven to preceding paragraph three, to a subjecthaving or at risk of having cancer.

In the method of the immediately preceding paragraph, the cancer may beone or more selected from the group consisting of: lung cancer,pancreatic, breast cancer, ovarian cancer, lung cancer, prostate cancer,head and neck cancer, gastric cancer, gastrointestinal cancer, coloncancer, esophageal cancer, cervical cancer, kidney cancer, biliary ductcancer, gallbladder cancer, and a hematologic malignancy.

A non-limiting example maintenance therapy for subjects that areundergoing or have undergone cancer therapy includes administering aneffective amount of the alpha polyglutamated tetrahydrofolatecomposition of any of preceding paragraphs from preceding paragraphthirteen to preceding paragraph five, to a subject that is undergoing orhas undergone cancer therapy.

A non-limiting example pharmaceutical composition may include any alphapolyglutamated tetrahydrofolate composition of Section IV.

A non-limiting example method for treating a disorder of the immunesystem may include administering an effective amount of the of the alphapolyglutamated tetrahydrofolate composition of any of precedingparagraphs from preceding paragraph fourteen to preceding paragraph six,to a subject having or at risk of having a disorder of the immunesystem.

A non-limiting example method for treating an infectious may includecomprises administering an effective amount of the of the alphapolyglutamated tetrahydrofolate composition of any of precedingparagraphs from preceding paragraph fifteen to preceding paragraphseven, to a subject having or at risk of having an infectious disease.

A non-limiting example method of delivering alpha polyglutamatedtetrahydrofolate to a tumor expressing a folate receptor on its surfacemay include administering a polyglutamated tetrahydrofolate compositionof any of preceding paragraphs from preceding paragraph sixteen topreceding paragraph eight, to a subject having the tumor in an amount todeliver a therapeutically effective dose of the alpha polyglutamatedtetrahydrofolate to the tumor.

A non-limiting example method of preparing a liposomal alphapolyglutamated tetrahydrofolate composition which includes alphapolyglutamated tetrahydrofolate composition of any of precedingparagraphs from preceding paragraph seventeen to preceding paragraphnine includes forming a mixture comprising: liposomal components; alphapolyglutamated tetrahydrofolate in solution; homogenizing the mixture toform liposomes in the solution; and processing the mixture to formliposomes containing the polyglutamated tetrahydrofolate.

A non-limiting example pharmaceutical composition includes an alphapolyglutamated tetrahydrofolate composition of any of precedingparagraphs from preceding paragraph eighteen to preceding paragraph ten.

Although the disclosure has been described with reference to varioussome embodiments, it should be understood that various modifications canbe made without departing from the spirit of the disclosure.Accordingly, the scope of the disclosure should be determined withreference to the appended claims, along with the full scope ofequivalents to which such claims are entitled. Throughout thisapplication, various publications are referenced by author name anddate, or by Patent No. or Patent Publication No. The disclosure of thesepublications are hereby incorporated in their entireties by referenceinto this application in order to more fully describe the state of theart as known to those skilled therein as of the date of the inventiondescribed and claimed herein. However, the citation of a referenceherein should not be construed as an acknowledgement that such referenceis prior art to the present invention.

Various new chemical entities, methods and equipment for making thesechemical entities are set forth below in the appended claims.

It is to be appreciated that the Detailed Description section, and notthe Summary and Abstract sections, is intended to be used to interpretthe claims. The Summary and Abstract sections may set forth one or morebut not all exemplary embodiments, of the present invention ascontemplated by the inventor(s), and thus, are not intended to limit thepresent invention and the appended claims in any way.

The disclosure of each of U.S. Appl. No. 62/627,703, filed Feb. 7, 2018;U.S. Appl. No. 62/627,714, filed Feb. 7, 2018; U.S. Appl. No.62/627,716, filed Feb. 7, 2018; U.S. Appl. No. 62/627,731, filed Feb. 7,2018; U.S. Appl. No. 62/627,741, filed Feb. 7, 2018; U.S. Appl. No.62/630,629, filed Feb. 14, 2018; U.S. Appl. No. 62/630,634, filed Feb.14, 2018; U.S. Appl. No. 62/630,637, filed Feb. 14, 2018; U.S. Appl. No.62/630,671, filed Feb. 14, 2018; U.S. Appl. No. 62/630,713, filed Feb.14, 2018; U.S. Appl. No. 62/630,728, filed Feb. 14, 2018; U.S. Appl. No.62/630,744, filed Feb. 14, 2018; U.S. Appl. No. 62/630,820, filed Feb.14, 2018; U.S. Appl. No. 62/630,825, filed Feb. 14, 2018; U.S. Appl. No.62/636,294, filed Feb. 28, 2018; U.S. Appl. No. 62/662,374, filed Apr.25, 2018; U.S. Appl. No. 62/702,732, filed Jul. 24, 2018; U.S. Appl. No.62/702,561, filed Jul. 24, 2018; U.S. Appl. No. 62/764,943, filed Aug.17, 2018; and U.S. Appl. No. 62/764,955, filed Aug. 17, 2018; is hereinincorporated by reference in its entirety.

1. A liposomal composition comprising an alpha polyglutamated alphatetrahydrofolate encapsulated by a liposome.
 2. The liposomalcomposition of claim 1, wherein the alpha polyglutamatedtetrahydrofolate is selected from the group consisting of: (a)polyglutamated 5-formyl-THF (e.g., polyglutamated [6S]-5-formyl-THF);(b) polyglutamated 10-formyl-THF (e.g., polyglutamated[6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF (e.g.,polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF(e.g., polyglutamated [6S]-5-methyl-THF); (e) polyglutamatedtetrahydrofolate (e.g., polyglutamated [6S]-Tetrahydrofolate THF); (f)polyglutamated 5,10-methylene-THF (e.g., polyglutamated[6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g.,polyglutamated [6S]-5-formimino-THF). 3.-6. (canceled)
 7. The liposomalcomposition of claim 1, wherein (a) two or more glutamyl groups have analpha carboxyl group linkage, (b) each of the glutamyl groups other thanthe glutamyl group of tetrahydrofolate has an alpha carboxyl grouplinkage, (c) two or more glutamyl groups have a gamma carboxyl grouplinkage, (d) at least one glutamyl group has both an alpha carboxylgroup linkage and a gamma carboxyl group linkage, (e) at least 2 of theglutamyl groups of the alpha polyglutamated tetrahydrofolate are in theL-form, (f) each of the glutamyl groups of the alpha polyglutamatedtetrahydrofolate is in the L-form, (g) at least 1 of the glutamyl groupsof the alpha polyglutamated tetrahydrofolate is in the D-form, (h) eachof the glutamyl groups of the alpha polyglutamated tetrahydrofolateother than the glutamyl group of tetrahydrofolate is in the D-form, or(i) at least 2 of the glutamyl groups of the alpha polyglutamatedtetrahydrofolate are in the L-form and at least 1 of the glutamyl groupsis in the D-form. 8.-9. (canceled)
 10. The liposomal composition ofclaim 1, wherein the polyglutamate is linear or branched. 11.-15.(canceled)
 16. The liposomal composition of claim 1, wherein theliposome comprises an alpha polyglutamated tetrahydrofolate containing4, 5, 2-10, 4-6, or more than 5, glutamyl groups. 17.-19. (canceled) 20.The liposomal composition of claim 1, wherein the liposome comprises analpha tetraglutamated tetrahydrofolate, alpha pentaglutamatedtetrahydrofolate, or alpha hexaglutamated tetrahydrofolate.
 21. Theliposomal composition of claim 1, wherein the liposome comprises a alphatetraglutamated tetrahydrofolate, alpha pentaglutamatedtetrahydrofolate, or alpha hexaglutamated alpha tetrahydrofolate. 22.(canceled)
 23. The liposomal composition of claim 1, wherein theliposome is pegylated.
 24. (canceled)
 25. The liposomal composition ofclaim 1, wherein the liposome has a diameter in the range of 20 nm to500 nm or 20 nm to 200 nm, or 80 nm to 120 nm.
 26. (canceled)
 27. Theliposomal composition of claim 1, wherein the liposome is formed fromliposomal components comprising: at least one of an anionic lipid and aneutral lipid; at least one selected from: DSPE; DSPE-PEG;DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; andcholesterol-maleimide; or at least one selected from: DSPE; DSPE-PEG;DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC. 28.-30.(canceled)
 31. The liposomal composition of claim 1, wherein one or moreliposomal components further comprises a steric stabilizer selectedfrom: polyethylene glycol (PEG); poly-L-lysine (PLL);monosialoganglioside (GM1); poly(vinyl pyrrolidone) (PVP);poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline);poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol;poly[N-(2-hydroxypropyl) methacrylamide]; amphiphilicpoly-N-vinylpyrrolidones; L amino-acid-based polymer; oligoglycerol,copolymer containing polyethylene glycol and polypropylene oxide,Poloxamer 188, and polyvinyl alcohol.
 32. (canceled)
 33. The liposomalcomposition of claim 1, wherein the steric stabilizer is PEG and the PEGhas a number average molecular weight (Mn) of 200 to 5000 daltons. 34.The liposomal composition of claim 1, wherein the liposome is anionic orneutral.
 35. The liposomal composition of claim 1, wherein the liposomehas a zeta potential that is less than or equal to zero, between 0 to−150 mV, or between −30 to −50 mV. 36.-37. (canceled)
 38. The liposomalcomposition of claim 1, wherein the liposome is cationic.
 39. Theliposomal composition of claim 1, wherein the liposome has an interiorspace comprising the alpha polyglutamated tetrahydrofolate and anaqueous pharmaceutically acceptable carrier comprising: a tonicity agentsuch as, dextrose, mannitol, glycerine, potassium chloride, sodiumchloride, at a concentration of greater than 1%; 1% to 50% trehalose; 1%to 50% dextrose; 5% dextrose suspended in an HEPES buffered solution; ora total concentration of sodium acetate and calcium acetate of between50 mM to 500 mM. 40.-47. (canceled)
 48. The liposomal composition ofclaim 1, wherein the liposome comprises less than 500,000, less than200,000 or between 10 to 100,000 molecules of the alpha polyglutamatedtetrahydrofolate or any range therein between.
 49. (canceled)
 50. Theliposomal composition of claim 1, which further comprises a targetingmoiety and wherein the targeting moiety has a specific affinity for asurface antigen on a target cell of interest.
 51. The liposomalcomposition of claim 50, wherein the targeting moiety is attached to oneor both of a PEG and the exterior of the liposome, optionally whereintargeting moiety is attached to one or both of the PEG and the exteriorof the liposome by a covalent bond.
 52. The liposomal composition ofclaim 50, wherein the targeting moiety is a polypeptide, an antibody oran antigen binding fragment of an antibody.
 53. (canceled)
 54. Theliposomal composition of claim 50, wherein the targeting moiety bindsthe surface antigen with an equilibrium dissociation constant (Kd) in arange of 0.5×10⁻¹⁰ to 10×10⁻⁶ as determined using surface plasmonresonance analysis.
 55. The liposomal composition of claim 54, whereinthe targeting moiety specifically binds one or more folate receptorsselected from the group consisting of: folate receptor alpha (FR-α),folate receptor beta (FR-β), and folate receptor delta (FR-δ).
 56. Theliposomal composition of claim 55, wherein the targeting moietycomprises one or more selected from the group consisting of: anantibody, a humanized antibody, an antigen binding fragment of anantibody, a single chain antibody, a single-domain antibody, abi-specific antibody, a synthetic antibody, a pegylated antibody, and amultimeric antibody.
 57. The liposomal composition of claim 50, whereinthe pegylated liposome comprises from 1 to 1000 or 30-200 targetingmoieties.
 58. The liposomal composition of claim 25, further comprisingone or more of an immunostimulatory agent, a detectable marker and amaleimide, wherein the immunostimulatory agent, the detectable marker orthe maleimide is attached to said PEG or the exterior of the liposome.59. (canceled)
 60. The liposomal composition of claim 58, wherein theimmunostimulatory agent is at least one selected from the groupconsisting of: a fluorescein; a fluorescein isothiocyanate (FITC); aDNP; a beta glucan; a beta-1,3-glucan; a beta-1,6-glucan; a Resolvin(e.g., a Resolvin D such as D_(n-6DPA) or D_(n-3DPA), a Resolvin E, or aT series Resolvin); and a Toll-like receptor (TLR) modulating agent suchas, an oxidized low-density lipoprotein (e.g. OXPAC, PGPC), and aneritoran lipid (e.g., E5564). 61.-63. (canceled)
 64. The liposomalcomposition of claim 1, which further comprises at least onecryoprotectant selected from the group consisting of mannitol,trehalose, sorbitol, and sucrose. 65.-66. (canceled)
 67. The liposomalcomposition of claim 1, which further comprises carboplatin and/orpembroluzumab.
 68. A pharmaceutical composition comprising the liposomalcomposition of claim
 1. 69.-71. (canceled)
 72. A method for treating orpreventing disease or chemical induced toxicity in a subject needingsuch treatment or prevention, the method comprising administering theliposomal composition of claim 1 to the subject.
 73. A method fortreating or preventing disease or chemical induced toxicity in a subjectneeding such treatment or prevention, the method comprisingadministering the liposomal composition of claim 1 to the subject.
 74. Amethod of killing a hyperproliferative cell that comprises contacting ahyperproliferative cell with the composition of claim
 1. 75. A method ofkilling a hyperproliferative cell that comprises contacting ahyperproliferative cell with the liposomal composition of claim
 1. 76.The method of claim 75, wherein the hyperproliferative cell is a cancercell, a mammalian cell, and/or a human cell.
 77. (canceled)
 78. A methodfor treating cancer that comprises administering an effective amount ofthe liposomal composition of claim 1 to a subject having cancer.
 79. Themethod of claim 78, wherein the liposomal composition is administered totreat or prevent cancer and wherein the cancer is selected from thegroup consisting of: a non-hematologic malignancy including such as forexample, lung cancer, pancreatic cancer, breast cancer, ovarian cancer,prostate cancer, head and neck cancer, gastric cancer, gastrointestinalcancer, colorectal cancer, esophageal cancer, cervical cancer, livercancer, kidney cancer, biliary duct cancer, gallbladder cancer, bladdercancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervoussystem cancer, and melanoma; and a hematologic malignancy such as forexample, a leukemia, a lymphoma and other B cell malignancies, myelomaand other plasma cell dyscrasias; lung cancer, breast cancer, coloncancer, pancreatic cancer, gastric cancer, bladder cancer, head and neckcancer, ovarian cancer, and cervical cancer; head and neck cancer,stomach cancer, osteosarcoma (e.g., osteosarcoma), Non-Hodgkin'slymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides(cutaneous T-cell lymphoma) choriocarcinoma, and chorioadenoma,nonleukemic meningeal cancer, soft tissue sarcoma (desmoid tumors,aggressive fibromatosis, bladder cancer, and central Nervous System(CNS) lymphoma; or colorectal cancer, breast cancer, ovarian cancer,lung cancer, head and neck cancer, pancreatic cancer, gastric cancer,and mesothelioma. 80.-82. (canceled)
 83. A method for treating cancerthat comprises administering an effective amount of the liposomalcomposition of claim 55 to a subject having a cancer cell that expresseson its surface a folate receptor bound by the targeting moiety. 84.(canceled)
 85. A maintenance therapy that comprises administering aneffective amount of the liposomal composition of any of claim 1 to asubject that is undergoing or has undergone cancer therapy.
 86. A methodfor treating a disorder of the immune system that comprisesadministering an effective amount of the composition of claim 1 to asubject having a disorder of the immune system.
 87. The method of claim86, wherein the disorder of the immune system is selected from:inflammation (e.g., acute and chronic), systemic inflammation,rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn disease,dermatomyositis/polymyositis, systemic lupus erythematosus, Takayasu,and psoriasis.
 88. A method for treating: (a) leukopenia that comprisesadministering an effective amount of the liposomal composition of claim1 to a subject having leukopenia. (b) a cardiovascular disease,metabolic disease, or a disease selected from: atherosclerosis,cardiovascular disease (CVD), coronary artery disease, myocardialinfarction, stroke, metabolic syndrome, a gestational trophoblasticdisease, and ectopic pregnancy; that comprises administering aneffective amount of the composition of claim 1 to a subject having thedisease; (c) an autoimmune disease, that comprises administering aneffective amount of the liposomal composition of claim 1 to a subjecthaving an autoimmune disease; (e) an inflammatory condition thatcomprises administering an effective amount of the liposomal compositionof claim 1 to a subject having inflammation, optionally wherein theinflammation is acute, chronic, and/or systemic inflammation; or (f) askin condition that comprises administering an effective amount of theliposomal composition of claim 1 to a subject having a skin condition.89. A method for treating an infectious disease that comprisesadministering an effective amount of the liposomal composition of claim1 to a subject having or at risk of having an infectious disease.
 90. Amethod of delivering alpha polyglutamated alpha tetrahydrofolate to atumor expressing a folate receptor on its surface, the methodcomprising: administering the liposomal composition of claim 55 to asubject having the tumor in an amount to deliver a therapeuticallyeffective dose of the polyglutamated alpha tetrahydrofolate to thetumor.
 91. A method of preparing a alpha tetrahydrofolate compositioncomprising the liposomal composition of claim 1, the method comprising:forming a mixture comprising: liposomal components and alphapolyglutamated tetrahydrofolate in solution; homogenizing the mixture toform liposomes in the solution; and processing the mixture to formliposomes containing alpha polyglutamated tetrahydrofolate.
 92. A methodof preparing the composition of claim 55, comprising the steps of:forming a mixture comprising: liposomal components and alphapolyglutamated tetrahydrofolate in a solution; homogenizing the mixtureto form liposomes in the solution; processing the mixture to formliposomes entrapping and/or encapsulating alpha polyglutamatedtetrahydrofolate; and providing a targeting moiety on a surface of theliposomes, the targeting moiety having specific affinity for at leastone of folate receptor alpha (FR-α), folate receptor beta (FR-β) andfolate receptor delta (FR-δ), optionally, wherein the processing stepincludes one or more steps of: thin film hydration, extrusion, in-linemixing, ethanol injection technique, freezing-and-thawing technique,reverse-phase evaporation, dynamic high pressure microfluidization,microfluidic mixing, double emulsion, freeze-dried double emulsion, 3Dprinting, membrane contactor method, and stirring, or wherein saidprocessing step includes one or more steps of modifying the size of theliposomes by one or more of steps of extrusion, high-pressuremicrofluidization, and/or sonication. 93.-94. (canceled)